具体实施方式
为了促进对本发明的理解,以下将参考某些实施方式,并且将使用特定语言来描述本发明。然而,应当理解的是,这些具体实施方式不意图限制本发明的范围。所描述的实施方式中的任何改变和进一步的修改,以及本发明的任何进一步应用,均为本领域技术人员通常会想到的。
重组蛋白
如本文所用,术语“重组蛋白”是指人工设计/构建的蛋白,而不是天然存在的蛋白质。本发明的“重组蛋白”中的“重组”不代表其生产方式,其仅用于表示“重组蛋白”并不天然存在。本发明的重组蛋白可以是表达的蛋白,可以是组装的蛋白。
任选地,本发明的重组蛋白包含高亲和靶向肿瘤的臂和低亲和阻断CD47与SIRPα相互作用的融合蛋白。
本文所使用的“高亲和靶向肿瘤”是指本发明的重组蛋白对肿瘤的结合亲和力高于或大致相当于现有技术中结合肿瘤的抗体类药物对肿瘤的结合亲和力,现有技术中结合肿瘤的抗体类药物对肿瘤的结合亲和力通常EC50在nM或pM水平。优选地,本发明的重组蛋白中,高亲和靶向肿瘤的臂所对应的抗体对肿瘤细胞上靶抗原的结合亲和力是低亲和阻断CD47与SIRPα相互作用的融合蛋白所对应的单体融合蛋白同二聚体对肿瘤细胞上CD47结合亲和力的至少6倍,任选地6倍、7倍、8倍、9倍、10倍、11倍、12倍、13倍、14倍、15倍,或更高的倍数值,或其间的任意数值。任选地,本发明的重组蛋白中,高亲和靶向肿瘤的臂所对应的抗体对肿瘤细胞上靶抗原的结合亲和力是低亲和阻断CD47与SIRPα相互作用的融合蛋白所对应的SIRPα-Fc同二聚体对肿瘤细胞上CD47结合亲和力的至少6倍,任选地6倍、7倍、8倍、9倍、10倍、11倍、12倍、13倍、14倍、15倍,或更高的倍数值,或其间的任意数值。
本文所使用的“低亲和阻断CD47与SIRPα相互作用”是指本发明的重组蛋白能够以较低的亲和力阻断CD47与SIRPα相互作用。优选地,本发明的重组蛋白中低亲和阻断CD47与SIRPα相互作用的融合蛋白对CD47的结合亲和力不高于含SIRPα胞外截短体的单体融合蛋白的同二聚体对CD47的结合亲和力。更优选地,不高于人源SIRPα-Fc融合蛋白对CD47的结合亲和力。
本文所述双特异性重组蛋白,当其高亲和靶向肿瘤的臂所对应的抗体对肿瘤细胞上靶抗原的结合亲和力是其阻断CD47与SIRPα相互作用的融合蛋白的臂所对应的单体融合蛋白同二聚体对肿瘤细胞上CD47结合亲和力的至少6倍时,即可实现显著提高具有调节巨噬细胞功能重组蛋白的肿瘤靶向饱和结合丰度和降低非肿瘤靶向的副作用的缺陷。
本文所使用的结合亲和力的测定方法是本领域技术人员熟知的,包括例如但不限于ELISA法和/或流式细胞术。
任选地,本发明所述信号调节蛋白α胞外截短体(含野生型胞外截短体及其非高亲和突变体胞外截短体)-Fc融合蛋白与半抗体可以通过改造抗体重链使形成异源二聚体,具体地,比如可以通过Knobs-into-holes连接和/或链间二硫键和/或盐键介导得到重组蛋白。
任选地,当“靶向肿瘤的臂”或“半抗体”或“左臂”或“半抗体结构”或“Ig分子单体”与“靶向CD47的臂”或“右臂”或“信号调节蛋白α截短体-Fc融合蛋白”或“SIRPα-Fc”通过knobs-into-holes连接得到重组蛋白时,所得重组蛋白的结构如图3所示。其中,左臂是肿瘤靶向的半抗体,包括但不限于抗5T4半抗体、抗AGS-16半抗体、抗ALK1半抗体、抗ANG-2半抗体、抗B7-H3半抗体、抗B7-H4半抗体、抗c-fms半抗体、抗c-Met半抗体、抗CA6半抗体、抗CD123半抗体、抗CD19半抗体、抗CD20半抗体、抗CD22半抗体、抗EpCAM半抗体、抗CD30半抗体、抗CD32b半抗体、抗CD37半抗体、抗CD38半抗体、抗CD40半抗体、抗CD52半抗体、抗CD70半抗体、抗CD74半抗体、抗CD79b半抗体、抗CD98半抗体、抗CEA半抗体、抗CEACAM5半抗体、抗CLDN18.2半抗体、抗CLDN6半抗体、抗CS1半抗体、抗CXCR4半抗体、抗DLL-4半抗体、抗EGFR半抗体、抗EGP-1半抗体、抗ENPP3半抗体、抗EphA3半抗体、抗ETBR半抗体、抗FGFR2半抗体、抗FN半抗体、抗FR-α半抗体、抗GCC半抗体、抗GD2半抗体、抗GPC-3半抗体、抗GPNMB半抗体、抗HER2半抗体、抗HER3半抗体、抗HLA-DR半抗体、抗ICAM-1半抗体、抗IGF-1R半抗体、抗IL-3R半抗体、抗LIV-1半抗体、抗MSLN半抗体、抗MUC16半抗体、抗MUC1半抗体、抗NaPi2b半抗体、抗结合素-4半抗体、抗Notch 2半抗体、抗Notch 1半抗体、抗PD-L1半抗体、抗PD-L2半抗体、抗PDGFR-α半抗体、抗PS半抗体、抗PSMA半抗体、抗SLTRK6半抗体、抗STEAP1半抗体、抗TEM1半抗体、抗VEGFR半抗体、抗CD25半抗体、抗CD27L半抗体、抗DKK-1半抗体、抗CSF-1R半抗体、抗MSB0010718C半抗体、抗BCMA半抗体、抗CD138半抗体。右臂是SIRPα(包括人源SIRPα野生型及其非高亲和突变体)胞外截短体通过与IgG抗体的铰链区和Fc区连接形成的融合蛋白。其中右臂Fc区为hole突变体时,对应左臂Fc区为knob突变体;右臂为knob突变体时,对应左臂Fc区为hole突变体。如本领域技术人员所知,Fc区还可以同时做多个holes和/或knobs突变。
下表1为重组蛋白的示例性分子结构。
表1重组蛋白的分子结构示例
序号 |
左臂 |
右臂 |
1 |
Anti-CD20-Fc1 |
D1-Fc2 |
2 |
Anti-CD20-Fc1 |
D1-D2-Fc2 |
3 |
Anti-CD20-Fc1 |
D1-D2-D3-Fc2 |
4 |
Anti-CD20-Fc1 |
D1<sup>m</sup>-Fc2 |
5 |
Anti-CD20-Fc1 |
D1<sup>m</sup>-D2-Fc2 |
6 |
Anti-PD-L1-Fc1 |
D1-Fc2 |
7 |
Anti-PD-L1-Fc1 |
D1-D2-Fc2 |
8 |
Anti-PD-L1-Fc1 |
D1-D2-D3-Fc2 |
9 |
Anti-PD-L1-Fc1 |
D1<sup>m</sup>-Fc2 |
10 |
Anti-PD-L1-Fc1 |
D1<sup>m</sup>-D2-Fc2 |
11 |
Anti-EGFR-Fc1 |
D1-Fc2 |
12 |
Anti-EGFR-Fc1 |
D1-D2-Fc2 |
13 |
Anti-EGFR-Fc1 |
D1-D2-D3-Fc2 |
14 |
Anti-EGFR-Fc1 |
D1<sup>m</sup>-Fc2 |
15 |
Anti-EGFR-Fc1 |
D1<sup>m</sup>-D2-Fc2 |
16 |
Anti-Her2-Fc1 |
D1-Fc2 |
17 |
Anti-Her2-Fc1 |
D1-D2-Fc2 |
18 |
Anti-Her2-Fc1 |
D1-D2-D3-Fc2 |
19 |
Anti-Her2-Fc1 |
D1<sup>m</sup>-Fc2 |
20 |
Anti-Her2-Fc1 |
D1<sup>m</sup>-D2-Fc2 |
D1m表示SIRPα胞外截短体D1的高亲和突变体;D1表示人源SIRPα野生型及其非高亲和突变体的胞外D1结构域;Fc表示野生型Fc区、Fc1表示具有hole或holes突变的Fc区、Fc2表示具有knob或knobs突变的Fc区
本发明重组蛋白的相应氨基酸序列和DNA序列见图17A-E并见本发明的序列表文件。
抗体
如本文所用,术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白,其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连,而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH),其后是多个恒定区。每条轻链的一端有可变区(VL),另一端有恒定区;轻链的恒定区与重链的第一个恒定区相对,轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
靶向肿瘤的臂
如本文所用,术语“靶向肿瘤的臂”或“半抗体”或“左臂”或“半抗体结构”或“Ig分子单体”指由抗体的一条轻链(L)和一条重链(H)组成的异二聚糖蛋白,是构成免疫球蛋白分子的基本结构,在本发明中可以互换使用;其分子量为对应抗体分子量的一半,约75000道尔顿,其中轻链通过一个共价二硫键与重链相连。重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH),其后是多个恒定区。每条轻链的一端有可变区(VL),另一端有恒定区;轻链的恒定区与重链的第一个恒定区相对,轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用,术语“靶向肿瘤的臂”、“半抗体”或“左臂”或“半抗体结构”或“Ig分子单体”可以是各种肿瘤靶向的IgG蛋白。肿瘤靶标分子包括但不限于5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGFR、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GD2、GPC-3、GPNMB、HER2、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch1、PD-L1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、BCMA、CD138。
“靶向肿瘤的臂”、“半抗体”或“左臂”或“半抗体结构”或“Ig分子单体”的Fc序列可以采用hole或holes突变体和/或knob或knobs突变体。
靶向CD47的臂
如本文所用,术语“靶向CD47的臂”或“右臂”或“信号调节蛋白α截短体-Fc融合蛋白”或“SIRPα-Fc”或“阻断CD47与SIRPα相互作用的融合蛋白”在本发明中可以互换使用。如本领域技术人员所知晓地,所述“靶向CD47的臂”或“右臂”或“信号调节蛋白α截短体-Fc融合蛋白”或“SIRPα-Fc”或“阻断CD47与SIRPα相互作用的融合蛋白”分子长度可变。任选地,可以通过SIRPα(包括人源SIRPα野生型及其非高亲和突变体)的胞外截短体与IgG1抗体的铰链区和Fc区连接,从而形成的多种分子长度不同的“靶向CD47的臂”或“右臂”或“信号调节蛋白α截短体-Fc融合蛋白”或“SIRPα-Fc”或“阻断CD47与SIRPα相互作用的融合蛋白”。IgG1可以是人源IgG1。
“靶向CD47的臂”或“右臂”或“信号调节蛋白α截短体-Fc融合蛋白”或“SIRPα-Fc”或“阻断CD47与SIRPα相互作用的融合蛋白”的Fc可以采用knob或knobs突变体和/或hole或holes突变体。
“靶向肿瘤的臂”或“半抗体”或“左臂”或“半抗体结构”或“Ig分子单体”与“靶向CD47的臂”或“右臂”或“信号调节蛋白α截短体-Fc融合蛋白”或“SIRPα-Fc”或“阻断CD47与SIRPα相互作用的融合蛋白”如本领域技术人员所知,可以通过改造Fc片段(区)使形成异源二聚体重组蛋白。具体地,本发明的重组蛋白,可以通过分子间作用力得到,也可以通过共价键例如链间二硫键得到,也可以通过盐键介导得到,也可以通过上述三种技术中的两种或三种的任意组合得到。任选地,本发明的重组蛋白是通过knobs-into-holes技术结合的。
knobs-into-holes技术
如本文所用,术语“knobs-into-holes技术”或“杵臼”技术或“隆突-入-空穴”技术或“纽扣”技术,是利用基因工程技术,在重链两个CH3结构域引入不同的突变来促使重链发生异源二聚化,一条重链上做一个钮(knob),在另一条重链上做一个扣(hole),然后两者优先咬合在一起形成不对称抗体(Ridgway JB,et al.'Knobs-into-holes'engineering ofantibody CH3domains for heavy chain heterodimerization.Protein Engineering,1996,9(7):617-621)。如本领域技术人员所知,一条重链上可以做多个钮(knob)和/或扣(hole),对应的,另一条重链上可以做多个扣(hole)和/或钮(knob)。
SIRPα
如本文所用,术语“SIRPα”是信号调节蛋白α(Signal Regulatory Proteinα),也称作CD172a。信号调节蛋白(SIRP)是一种穿膜糖蛋白,包括三个家族成员,SIRPα(CD172a)、SIRPβ(CD172b)、SIRPγ(CD172g)。这三个成员具有相似的膜外端,但不同的膜内区。膜外端均含有三个免疫球蛋白(Ig)样区域,其中第一个区域属于IgV区,第二、三个区域属于IgC区。SIRPα(CD172a)的膜内区含有两个抑制性信号域,可传递抑制信号,抑制细胞的相应功能。SIRPβ(CD172b)和SIRPγ(CD172g)膜内区很短,没有信号传递区域,但SIRPβ(CD172b)可通过适配器蛋白(Adaptor protein,如DAP12)传递激活信号。SIRP蛋白主要表达在巨噬细胞
树突状细胞(DC)、及神经元细胞。本文特指人源SIRPα野生型及其CD47非高亲和突变体。
SIRPα胞外截短体
“胞外截短体”是指针对具有跨膜功能的蛋白质。“SIRPα胞外截短体”,如本文所用,指人源SIRPα野生型及其CD47非高亲和突变体,选择性地截选其全部或部分位于细胞膜外部空间的氨基酸序列。
如本文所用,术语“D1、D2、D3”指SIRPα胞外的三个Ig样结构域,从蛋白质的氨基端开始依次为D1结构域(Ig可变区样结构域,IgV区)、D2结构域(Ig恒定区样结构域,IgC区)和D3结构域(Ig恒定区样结构域,IgC区)(Lee WY,et al.The Role of cis Dimerization ofSignal Regulatory Proteinα(SIRPα)in Binding to CD47.J Biol Chem,2010,285(49):37953-37963)。
SIRPα-Fc融合蛋白
如本文所用,术语“SIRPα-Fc融合蛋白”指包含SIRPα胞外截短体、接头序列和Fc区的融合蛋白。上述序列中所包含的接头序列和/或Fc区可根据本领域技术人员所熟知的方式或常用接头序列和/或Fc区任意替换。
为了避免糖基化的影响,本发明对D1进行了由天冬酰胺到丙氨酸的突变(参考文献:Lee W Y,et al.Novel Structural Determinants on SIRPαthat Mediate Bindingto CD47.Journal of Immunology,2007,179(11):7741-7750)。
本发明的D1、D2、D3还包括相应的接头序列。
接头序列
如本文所用,术语“接头序列”是指连接SIRPα胞外截短体和结合序列的氨基酸序列,任选地,为IgG抗体的铰链区,任选地,包含铰链区与IgG重链CH1结构域。上述序列中所包含的接头序列或铰链区序列可根据本领域技术人员所熟知的方式或常用接头序列或铰链区序列任意替换。
结合序列
如本文所用,术语“结合序列“是指使高亲和靶向肿瘤的臂与低亲和阻断CD47与SIRPα相互作用的融合蛋白相结合的序列,任选地,所述结合序列包含铰链区和Fc区,更任选地,所述Fc区包含knob或knobs和/或hole或holes突变。上述序列中所包含的结合序列或铰链区或Fc区序列可根据本领域技术人员所熟知的方式或常用结合序列或铰链区或Fc区序列任意替换。
CD47
CD47是一种穿膜糖蛋白,属于免疫球蛋白超家族成员,在包括红细胞在内的几乎所有细胞表面表达。CD47的配体包括黏附因子(integrin)、凝血酶敏感蛋白1(thrombospondin-1)、以及信号调节蛋白(SIRP)。CD47具有多种生物学功能,包括细胞迁移、T细胞、树突状细胞活化、轴突发育等。除此之外,CD47通过与SIRPα相互作用可抑制巨噬细胞的吞噬作用。CD47以这种方式传递一种所谓的“不要吃我”(“Don't eat me”)信号,可以保护红细胞、B细胞、T细胞等正常细胞不被巨噬细胞吞噬。
Ofa
如本文所用,术语“Ofa”、“Ofatumumab”、“Anti-CD20(Ofatumumab)”在本发明中可以互换使用,表示抗CD20抗体Ofatumumab。
Obi
如本文所用,术语“Obi”、“Obinutuzumab”、“Anti-CD20(Obinutuzumab)”在本发明中可以互换使用,表示抗CD20抗体Obinutuzumab。
Hu5F9-G4
如本文所用,术语“Anti-CD47mAb”、“抗CD47抗体”、“Hu5F9-G4”在本发明中可以互换使用,表示抗CD47抗体Hu5F9-G4。
Anti-EGFR mAb
如本文所用,术语“Anti-EGFR mAb”、“JMT101”在本发明中可以互换使用,表示抗EGFR抗体JMT101。JMT101为人源化抗EGFR单克隆抗体,参见ZL201210406288.3专利BA03。
曲妥珠单抗
如本文所用,术语“曲妥珠单抗”、“Trastuzumab”、“Anti-Her2(T)mAb”、“Herceptin”在本发明中可以互换使用,表示抗Her2抗体Trastuzumab。
帕妥珠单抗
如本文所用,术语“帕妥珠单抗”、“Pertuzumab”、“Anti-Her2(P)mAb”、“Perjeta”在本发明中可以互换使用,表示抗Her2抗体Pertuzumab。
Atezolizumab
如本文所用,术语“Tecentriq”、“Atezolizumab”在本发明中可以互换使用,表示抗PD-L1抗体Atezolizumab。
SIRPαD1-Fc
如本文所用,术语“SIRPαD1-Fc”、“D1-Fc”在本发明中可以互换使用,表示单链融合蛋白SIRPαD1-Fc的二聚体。
Ofa-Fc1
表示具有hole突变的Fc区的Ofatumumab半抗体。
Anti-Her2(T)-Fc1
表示具有hole突变的Fc区的Transtuzumab半抗体。
Anti-Her2(P)-Fc1
表示具有hole突变的Fc区的Pertuzumab半抗体。
Anti-EGFR-Fc1
表示具有hole突变的Fc区的抗EGFR半抗体。
D1-Fc2
表示包含SIRPαD1结构域胞外截短体和具有knob突变的Fc区的融合蛋白。
D1-D2-Fc2
表示包含SIRPαD1和D2结构域胞外截短体和具有knob突变的Fc区的融合蛋白。
D1-D2-D3-Fc2
表示包含SIRPαD1、D2和D3结构域胞外截短体和具有knob突变的Fc区的融合蛋白。
治疗
如本文所用,术语“治疗”、“疗法”和“医治”可以互换使用。术语“治疗”包括控制疾病、病症、病况的进展和相关症状,优选减少疾病、病症、病况或减轻疾病、病症、病况的一种或多种症状的影响。此术语包括治愈疾病或完全消除症状。此术语包括症状得到缓解。此术语还包括但不限于非治愈性的姑息性治疗。术语“治疗”包括给受试者施用治疗有效量的包含本发明的重组蛋白或融合蛋白的药物组合物,以预防或延迟、减轻或缓解疾病、病症、病况的进展或疾病、病症、病况的一种或多种症状的影响。
施用
如本文所用,术语“施用”是指将治疗有效量的包含本发明的重组蛋白或融合蛋白的药物组合物递送至受试者。施用可以全身施用也可以局部施用。施用可以通过施用装置进行,例如注射器。施用方式包括但不限于包埋、鼻吸、喷雾、注射等。施用途径包括吸入、鼻内、口服、静脉内、皮下或肌内施用等。
表2-1序列名称和序列编号的对应关系
表2-2重组蛋白和序列的对应关系
实施例1表达载体的构建
按照所设计的分子结构,将各组成部分氨基酸序列前后拼接在一起,根据中国仓鼠(Cricetulus griseus)对密码子的偏爱性,设计得到最优DNA编码序列,排除基因克隆操作等需要使用的酶切位点,然后在该序列5’端依次添加克隆位点、Kozak序列和信号肽编码序列,在该序列3’端依次添加终止密码子和克隆位点,如图1所示。
进行全基因合成,利用5’端和3’端克隆位点将全基因定向克隆到表达载体pCHO-TE2(购自Thermo Fisher)相应的克隆位点之间,经测序验证正确之后,即可获得表达质粒。5’端和3’端所使用的克隆位点均为EcoRV和PacI位点。图2为表达载体pCHO-TE2的质粒图谱。
实施例2表达质粒的制备、细胞转染以及靶蛋白的表达、纯化
表达质粒的制备
将含有表达质粒的甘油菌(1mL的含有表达质粒的大肠杆菌菌液中加入0.5mL60%的无菌甘油溶液充分混匀)按1:1000的比例接种进液体LB培养基中。在37℃,220rpm,摇床培养16小时后离心收集菌体。使用无内毒素质粒大提试剂盒(DP117,购自天根生化科技(北京)有限公司),按照试剂盒说明书提供的标准流程抽提得到表达质粒。
细胞转染、蛋白表达
将所得表达质粒用0.22μm滤膜过滤后,吸取3mg质粒(其中,产物为典型抗体分子,其轻链、重链表达质粒比例为1:1(摩尔比);产物为重组蛋白,其轻链、重链、右臂表达质粒的比例为1:1:1(摩尔比),具体见表3)加入到50mL Opti MEM I Reduced Serum Medium(购自GIBCO)中混匀。吸取6mg转染试剂聚醚酰亚胺(Polyetherimide,PEI,购自Polysciences,以1mg/mL浓度溶于无菌超纯水中)加入到50mL Opti MEM I Reduced Serum Medium中混匀。将得到的PEI溶液加入含有质粒的Opti MEM I Reduced Serum Medium溶液中,混匀。室温静置15分钟后将质粒和PEI的混合物缓慢均匀加入体积为1L,细胞密度为3×106个细胞/mL的宿主细胞CHO-S(购自Thermo Fisher)悬液中,置于37℃、5%CO2培养箱培养中培养。4小时后,加入相当于初始体积7%的补料培养基(该补料培养基配方为每升水中溶解有80gCD Efficient FeedC AGT(购自Gibco)、75g 5×00483(购自Kerry))。将培养温度降低至33℃,培养6天后收获。将细胞悬液在10000g、10℃条件下离心30分钟,将离心所得上清即细胞培养收获液用于目的蛋白纯化。
蛋白纯化
以下方法以Ofa-Fc1-D1-Fc2为例,蛋白A亲和捕获。
将上述细胞培养收获液在10000rpm条件下离心30min去除细胞以及其碎片,然后上样蛋白A亲和柱(货号:17-5438-02,GE Healthcare),洗脱收获目标蛋白。SDS-PAGE检测蛋白纯度。
蛋白A纯化方法为本领域技术人员熟知的常规蛋白纯化方法,具体试验方法可参考GE Healthcare蛋白A产品使用说明及GE抗体纯化手册。
SIRPαD1-Fc、Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2四种蛋白的理论分子量分别为:37.8kD、110.7kD、121.7kD和131.4kD。SDS-PAGE蛋白电泳检测结果如图4A和图4B所示。
蛋白电泳(SDS-PAGE):结果显示(图4A和图4B),各泳道的目标蛋白都得到了有效表达和纯化,其中Ofa-Fc1-D1-Fc2(图4A泳道11)、Ofa-Fc1-D1-D2-Fc2(图4B泳道1)、Ofa-Fc1-D1-D2-D3-Fc2(图4A泳道12)可见不同程度的左臂二聚体(Ofa-Fc1-Ofa-Fc1)、右臂二聚体(SIRPα-Fc2)和/或多聚体。
表3表达质粒比例
备注:D1m表示SIRPα胞外截短体D1的高亲和突变体;D1表示人源SIRPα野生型及其非高亲和突变体的胞外D1结构域;Fc为野生型Fc区、Fc1为具有hole或holes突变的Fc区、Fc2为具有knob或knobs突变的Fc区
实施例3靶标亲和力、靶标竞争结合活性检测
1、CD47、CD20、EGFR、Her2靶标亲和力检测方法
重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2对靶标CD47以及CD20的结合亲和力通过ELISA法和/或流式细胞术进行测定。以下方法以Ofa-Fc1-D1-Fc2为例,适用于左臂为CD20靶点的重组蛋白的检测。
重组蛋白Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1m-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1m-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1m-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1m-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2对靶标CD47以及Her2的结合亲和力通过ELISA法和/或流式细胞术进行测定。以下方法以Anti-Her2(T)-Fc1-D1-Fc2为例,适用于左臂为Her2靶点的重组蛋白的检测。
重组蛋白Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1m-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1m-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2对靶标CD47以及EGFR的结合亲和力通过ELISA法和/或流式细胞术进行测定。以下方法以Anti-EGFR-Fc1-D1-Fc为例,适用于左臂为EGFR靶点的重组蛋白为例进行检测。
重组蛋白Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2对靶标CD47以及PD-L1的结合亲和力通过ELISA法和/或进行测定。以下方法以Anti-PD-L1(Ate)-Fc1-D1-Fc2为例,适用于左臂为PD-L1靶点的重组蛋白为例进行检测。
ELISA检测Ofa-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-Fc2对靶标CD47的亲和力:
用100μl 1μg/ml CD47-His(12283-H08H-200,Sino Biological)包被酶标板(货号:9018,Corning)并4℃放置过夜;PBST溶液(含0.1%浓度的Tween20的PBS)清洗后,用PBS+1%BSA对酶标板室温封闭2小时;清洗后,向包被板中分别加入稀释的Ofa-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-Fc2(1000ng/ml起始,2.5倍稀释,共11个点),每孔100μl,25℃孵育1小时;弃样品并用PBST溶液清洗三次;加入100μl稀释好的小鼠抗人IgG Fc-HRP(1:10000)(Ab7499,abcam)后,25℃孵育1小时;弃溶液并用PBST溶液清洗三次;加入TMB(P0209,beyotime)避光显色约20分钟后,用H2SO4终止反应,在酶标仪上读取OD(450-650nm)值。
试验结果显示,抗CD47抗体Hu5F9-G4、SIRPαD1-Fc、Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1m-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1m-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2均分别能与CD47结合;Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2对CD47的结合亲和力略弱于抗CD47抗体Hu5F9-G4和/或SIRPαD1-Fc对CD47的结合亲和力。
以上试验数据证明,本发明的重组蛋白在蛋白水平能够特异性靶向肿瘤细胞CD47抗原,且与CD47的结合亲和力不高于SIRPαD1-Fc融合蛋白对CD47蛋白的结合亲和力;本发明的重组蛋白能够降低或避免抗CD47抗体治疗产生的红细胞凝集、贫血和/或SIRPα高亲和突变体治疗产生的非肿瘤靶细胞杀伤等副作用(Petrova PS,et al.TTI-621(SIRPαFc):ACD47-Blocking Innate Immune Checkpoint Inhibitor with Broad AntitumorActivity and Minimal Erythrocyte Binding.Clin Cancer Res,2017,23(4):1068-1079)。
例如,如图5所示,除抗CD20抗体Ofatumumab以及抗EGFR抗体JMT101不能结合CD47外,抗CD47抗体Hu5F9-G4、SIRPαD1-Fc、Ofa-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-Fc2均能与CD47结合,但由于Ofa-Fc1-D1-Fc2和Anti-EGFR-Fc1-D1-Fc2仅有右臂能与CD47抗原结合等原因,其亲和力(EC50=52.57ng/mL/EC50=93.86ng/mL)弱于抗CD47抗体(EC50=5.439ng/mL)和/SIRPαD1-Fc(EC50=6.118ng/mL)。
流式细胞术检测Ofa-Fc1-D1-Fc2对靶标CD47的亲和力:
A431细胞(人表皮癌细胞),收集生长良好的细胞并计数,离心并用PBS+2%FBS(购自Gibco)重悬细胞至3×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入9个稀释度的Ofa-Fc1-D1-Fc2(从15000ng/ml起始,5倍梯度稀释,共9个浓度),并将96孔板在冰箱中4℃孵育1小时;用PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(Accuri C6,BD)检测荧光值。
由于A431细胞不表达CD20抗原,不能与Ofatumumab、Obinutuzumab结合,因此可以在细胞水平上用A431细胞评价Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2与CD47的结合亲和力。
试验结果显示,除抗CD20抗体Ofatumumab、Obinutuzumab不能结合A431外,抗CD47抗体Hu5F9-G4、SIRPαD1-Fc、Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2均能与A431细胞结合;Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2对CD47的结合亲和力略弱于抗CD47抗体和/或SIRPαD1-Fc对CD47的结合亲和力,与ELISA的数据趋势相一致。
以上试验数据证明,本发明的重组蛋白在细胞水平能够特异性靶向肿瘤细胞CD47抗原,且与CD47的结合亲和力不高于SIRPαD1-Fc融合蛋白对CD47的结合亲和力;本发明的重组蛋白能够降低或避免抗CD47抗体治疗产生的红细胞凝集、贫血和/或SIRPα高亲和突变体治疗产生的非肿瘤靶细胞杀伤等副作用。
例如,如图6所示,抗CD47抗体Hu5F9-G4、SIRPαD1-Fc和Ofa-Fc1-D1-Fc2均能与A431细胞结合。具体地,Ofa-Fc1-D1-Fc2的亲和力略弱于抗CD47抗体和/或SIRPαD1-Fc,与ELISA的数据趋势相一致。
流式细胞术检测Ofa-Fc1-D1-Fc2对靶标CD20的亲和力:
Raji细胞(人B细胞淋巴瘤)(购自上海中科院细胞库),收集生长良好的细胞并计数,离心并用PBS+2%FBS重悬细胞至3×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并加入100μL PBS+2%FBS(对照组)或1.5μg/mL的抗CD47抗体Hu5F9-G4(Fab)2(实验组)(经胃蛋白酶切除Fc,试剂盒:Thermo Fisher,44988),4℃孵育1小时;PBS+2%FBS清洗后,分别加入7个稀释度的Ofa-Fc1-D1-Fc2、Hu5F9-G4、SIRPαD1-Fc(从6250ng/ml起始,4倍梯度稀释,共7个浓度,图7中为转化后的摩尔浓度),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(AccuriC6,BD)检测荧光值。
试验结果显示,抗CD47抗体Hu5F9-G4(Fab)2可有效地阻断抗CD47抗体Hu5F9-G4和/或SIRPαD1-Fc与Raji细胞上的CD47的结合;但Hu5F9-G4(Fab)2对CD47抗原的封闭作用并没有显著抑制Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2与Raji细胞的结合。
以上试验数据证明,在肿瘤细胞表面CD47抗原被封闭、SIRPα-CD47相互结合被阻断的情况下,本发明的重组蛋白仍然能依靠其左臂与肿瘤细胞上对应的抗原特异性结合,且左臂亲和力并未因右臂结合被阻断而受到显著影响。
例如,如图7所示,抗CD47抗体Hu5F9-G4(Fab)2可有效地阻断抗CD47抗体Hu5F9-G4和/或SIRPαD1-Fc与Raji细胞上的CD47的结合,但Hu5F9-G4(Fab)2对CD47抗原的封闭作用并没有显著抑制Ofa-Fc1-D1-Fc2与Raji细胞的结合,表明Ofa-Fc1-D1-Fc2在SIRPα-CD47相互结合被阻断后,仍然能依靠其左臂(抗CD20半抗体)与Raji细胞上的CD20抗原特异性结合,且亲和力并未因右臂结合被阻断而受到显著影响。
流式细胞术检测与靶标CD20和CD47的双特异性结合情况:
Raji细胞(人B细胞淋巴瘤)(购自上海中科院细胞库),收集生长良好的细胞并计数,离心并用PBS+2%FBS重悬细胞至3×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入12个稀释度的Ofa-Fc1-D1-Fc2、Ofatumumab、Hu5F9-G4、SIRPαD1-Fc(50000ng/ml、25000ng/ml、6250ng/ml,然后4倍梯度稀释,共12个浓度,图8A中为转化后的摩尔浓度),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(Accuri C6,BD)检测荧光值。
由于Raji细胞表面同时表达CD20和CD47抗原,因此,抗CD20抗体Ofatumumab、Obinutuzumab、抗CD47抗体Hu5F9-G4和SIRPαD1-Fc均能与Raji细胞特异性结合,但所能达到的最大平均荧光强度各不相同。
试验结果显示,Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1m-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1m-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1m-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2也能与Raji细胞结合,且具有更高的最大平均荧光强度。
以上试验数据证明,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与抗CD20抗体Ofatumumab、Obinutuzumab和/或抗CD47抗体Hu5F9-G4和/或SIRPαD1-Fc相比,能与肿瘤细胞特异性结合,且表现出显著的分子数量优势。优选的,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与肿瘤细胞的饱和结合丰度大于抗CD20抗体和SIRPαD1-Fc与肿瘤细胞饱和结合丰度之和。
表4抗体/重组蛋白和Raji细胞结合的最大平均荧光强度和EC50(nM)
例如,如图8A和表4所示,抗CD20抗体Ofatumumab、抗CD47抗体Hu5F9-G4和SIRPαD1-Fc均能与Raji细胞特异性结合,但所能达到的最大平均荧光强度各不相同;同时Ofa-Fc1-D1-Fc2也能与Raji细胞结合,且具有更高的最大平均荧光强度,提示Raji细胞在相同的过饱和蛋白样品浓度环境下,能特异性结合的Ofa-Fc1-D1-Fc2蛋白分子数量分别显著高于抗CD20抗体Ofatumumab或抗CD47抗体Hu5F9-G4或SIRPαD1-Fc的分子数量,高于抗CD20抗体Ofatumumab与抗CD47抗体Hu5F9-G4分子数量之和,和高于抗CD20抗体Ofatumumab与SIRPαD1-Fc分子数量之和。
如本领域技术人员所知,以上试验结果提示,与抗CD47抗体或SIRPαD1-Fc与其他抗肿瘤靶向治疗抗体的联合使用相比,使用本发明能够与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白可以更多地结合到肿瘤细胞,从而获得更显著的抗肿瘤功效。
流式细胞术检测与靶标Her2和CD47的双特异性结合情况:
SKBR-3细胞(人乳腺癌细胞)(购自上海中科院细胞库),收集生长良好的细胞并计数,离心并用PBS+2%FBS重悬细胞至2×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入各自10个稀释度的Anti-Her2(T)-Fc1-D1-Fc2、Trastuzumab和Hu5F9-G4(433.2nM起始、4倍梯度稀释,共10个点),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(Accuri C6,BD)检测荧光值。
由于SKBR-3细胞表面同时表达Her2和CD47抗原,因此,抗Her2抗体Trastuzumab、Pertuzumab、抗CD47抗体Hu5F9-G4和SIRPαD1-Fc均能与SKBR-3细胞特异性结合,但所能达到的最大平均荧光强度各不相同。
试验结果显示,Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1m-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1m-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1m-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1m-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1m-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1m-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1m-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1m-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2也能与SKBR-3细胞结合,且具有更高的最大平均荧光强度。
以上试验数据证明,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与抗Her2抗体Trastuzumab、Pertuzumab和/或抗CD47抗体Hu5F9-G4和/或SIRPαD1-Fc相比,能与肿瘤细胞特异性结合,且表现出显著的分子数量优势。优选的,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与肿瘤细胞的饱和结合丰度大于对应抗Her2抗体和SIRPαD1-Fc与肿瘤细胞饱和结合丰度之和。
表5抗体/重组蛋白和SKBR-3细胞结合的最大平均荧光强度和EC50(nM)
例如,如图8B和表5所示,抗Her2抗体Trastuzumab和抗CD47抗体Hu5F9-G4均能与SKBR-3细胞特异性结合,但所能达到的最大平均荧光强度各不相同;同时Anti-Her2(T)-Fc1-D1-Fc2也能与SKBR-3细胞结合,且具有更高的最大平均荧光强度,提示SKBR-3细胞在相同的过饱和蛋白样品浓度环境下,能特异性结合的Anti-Her2(T)-Fc1-D1-Fc2蛋白分子数量分别显著高于抗Her2抗体Trastuzumab或抗CD47抗体Hu5F9-G4的分子数量,且高于抗Her2抗体Trastuzumab和抗CD47抗体Hu5F9-G4分子数量之和。
如本领域技术人员所知,以上试验结果提示,与抗CD47抗体或SIRPαD1-Fc与其他抗肿瘤靶向治疗抗体的联合使用相比,使用本发明的能够与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白可以更多地结合到肿瘤细胞,从而获得更显著的抗肿瘤功效。
流式细胞术检测与靶标EGFR和CD47的双特异性结合情况:
A431细胞(人表皮癌细胞)(购自中国医学科学院基础医学研究所),收集生长良好的细胞并计数,离心并用PBS+2%FBS重悬细胞至2×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入各自11个稀释度的Anti-EGFR-Fc1-D1-Fc2、JMT101、SIRPαD1-Fc和Hu5F9-G4(216.6nM起始,4倍梯度稀释,共11个浓度),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(AccuriC6,BD)检测荧光值。
由于A431细胞表面同时表达EGFR和CD47抗原,因此,抗EGFR抗体JMT101、抗CD47抗体Hu5F9-G4、SIRPαD1-Fc均能与A431细胞特异性结合,但所能达到的最大平均荧光强度各不相同。
试验结果显示,Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1m-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1m-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2也能与A431细胞结合,且具有更高的最大平均荧光强度。
以上试验数据证明,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与抗EGFR抗体JMT101和/或抗CD47抗体Hu5F9-G4和/或SIRPαD1-Fc相比,能与肿瘤细胞特异性结合,且表现出显著的分子数量优势。优选的,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与肿瘤细胞的饱和结合丰度大于对应抗EGFR抗体和SIRPαD1-Fc与肿瘤细胞饱和结合丰度之和。
表6抗体/重组蛋白和A431细胞结合的最大平均荧光强度和EC50(nM)
例如,如图8C和表6所述,抗EGFR抗体JMT101、抗CD47抗体Hu5F9-G4、SIRPαD1-Fc均能与A431细胞特异性结合,但所能达到的最大平均荧光强度各不相同,且EC50分别为EC50(JMT101)=0.598nM、EC50(SIRPαD1-Fc)=3.677nM、EC50(Hu5F9-G4)=0.865nM,可见JMT101与A431细胞的结合亲和力是SIRPαD1-Fc与A431细胞的结合亲和力6倍以上;同时Anti-EGFR-Fc1-D1-Fc2也能与A431细胞结合,且具有更高的最大平均荧光强度,提示A431细胞在相同的过饱和蛋白样品浓度环境下,能特异性结合的Anti-EGFR-Fc1-D1-Fc2蛋白分子数量分别显著高于抗EGFR抗体JMT101或SIRPαD1-Fc或抗CD47抗体Hu5F9-G4的分子数量,且高于抗EGFR抗体JMT101和SIRPαD1-Fc的分子数量之和。
如本领域技术人员所知,以上试验结果提示,与抗CD47抗体或SIRPαD1-Fc与其他抗肿瘤靶向治疗抗体的联合使用相比,使用本发明的能够与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白可以更多地结合到肿瘤细胞,从而获得更显著的抗肿瘤功效。
流式细胞术检测与靶标PD-L1和CD47的双特异性结合情况:
NCI-H441细胞(人肺腺癌细胞)(购自北京北纳创联生物技术研究院),用10ng/mlhIFN-γ(BD,货号:554616)刺激2×107个细胞18h,消化收集细胞并计数,离心并用PBS+2%FBS重悬细胞至3×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入各自12个稀释度的Anti-PD-L1(Ate)-Fc1-D1-Fc2和Atezolizumab(433.2nM、216.6nM,然后4倍梯度稀释,共12个浓度),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(Accuri C6,BD)检测荧光值。
由于NCI-H441细胞表面同时表达PD-L1和CD47抗原,因此,抗PD-L1抗体Atezolizumab、13G4、12A4、抗CD47抗体Hu5F9-G4、SIRPαD1-Fc能与NCI-H441细胞特异性结合。
试验结果显示,Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2也均能与NCI-H441细胞结合,且具有更高的最大平均荧光强度。
以上试验数据证明,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与抗PD-L1抗体Atezolizumab、13G4、12A4相比,能与肿瘤细胞特异性结合,且表现出一定的分子数量优势。例如,在相同的过饱和蛋白样品浓度环境下,本发明的重组蛋白与抗PD-L1抗体Atezolizumab相比,能更多地结合到肿瘤细胞,表现出显著的分子数量优势。
表7抗体/重组蛋白和NCI-H441细胞结合的最大平均荧光强度和EC50(nM)
例如,如图8D和表7所示,抗PD-L1抗体Atezolizumab能与NCI-H441细胞特异性结合;同时Anti-PD-L1(Ate)-Fc1-D1-Fc2也能与NCI-H441细胞结合,且具有更高的最大平均荧光强度,提示NCI-H441细胞在相同的过饱和蛋白样品浓度环境下,能特异性结合的Anti-PD-L1(Ate)-Fc1-D1-Fc2蛋白分子数量高于抗PD-L1抗体Atezolizumab的分子数量。EC50分别为EC50(Atezolizumab)=0.2006nM、EC50(SIRPαD1-Fc)=1.643nM、EC50(Hu5F9-G4)=0.3865nM,可见Atezolizumab与NCI-H441细胞的结合亲和力是SIRPαD1-Fc与NCI-H441细胞的结合亲和力6倍以上。
如本领域技术人员所知,以上试验结果提示,与抗CD47抗体与其他抗肿瘤靶向治疗抗体的联合使用相比,使用本发明的能够与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白可以更多地结合到肿瘤细胞,从而获得更显著的抗肿瘤功效。
2、靶标竞争结合活性检测
以下方法以Ofa-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-Fc2为例,通过ELISA法对靶标CD47与SIRPα结合的竞争结合活性进行测定。
ELISA检测Ofa-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-Fc2的竞争结合活性:
用100μl 1μg/ml CD47-His(12283-H08H-200,Sino Biological)包被酶标板(9018,Corning)并4℃放置过夜;PBST清洗后,用PBS+1%BSA对酶标板室温封闭2h;清洗后,向包被板中分别加入稀释的不同浓度(1000ng/ml起始,3倍梯度稀释,共11个浓度)的Ofa-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-Fc2和生物素标记的SIRPαD1-Fc(生物素标记试剂盒,21925,Thermo,加样浓度为100ng/ml)混合液,每孔100μl,25℃孵育1小时;弃去样品并用PBST溶液清洗三次;加入100μl稀释好的链霉亲和素-HRP(1:10000)(ML-0437P-HRP,ZI501-1研域化学试剂)后,25℃孵育1小时;弃溶液并用PBST溶液清洗三次;加入TMB(P0209,beyotime)避光显色约20分钟后,用H2SO4终止反应,在酶标仪上读取OD(450-650nm)值。
试验结果显示,抗CD47抗体Hu5F9-G4、SIRPαD1-Fc、Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2均能不同程度地与生物素标记的SIRPαD1-Fc竞争性结合CD47抗原,发挥竞争结合活性。
例如,如图9所示,抗CD47抗体Hu5F9-G4、SIRPαD1-Fc、Ofa-Fc1-D1-Fc2和Anti-EGFR-Fc1-D1-Fc2均能不同程度地与生物素标记的SIRPαD1-Fc竞争性结合CD47抗原,发挥竞争结合活性;Ofa-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-Fc2与CD47的竞争性结合力分别弱于抗CD47抗体Hu5F9-G4或SIRPαD1-Fc,这与本实施例上文所述的亲和力结果相一致。
实施例4重组蛋白早期体外免疫安全性评价实验
重组蛋白Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1m-D2-Fc2、Anti-EGFR-Fc1-D1m-Fc2、Anti-EGFR-Fc1-D1m-D2-Fc2、Anti-Her2(T)-Fc1-D1m-Fc2、Anti-Her2(T)-Fc1-D1m-D2-Fc2、Anti-Her2(P)-Fc1-D1m-Fc2、Anti-Her2(P)-Fc1-D1m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-D2-Fc2早期体外免疫安全性评价实验,以下方法以Ofa-Fc1-D1m-Fc2为例,适用于右臂含SIRPα胞外截短体高亲和突变体的重组蛋白的检测。
Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2早期体外免疫安全性评价实验,以下方法以Ofa-Fc1-D1-Fc2为例,适用于右臂含SIRPα胞外截短体的重组蛋白的检测。
流式细胞术检测双抗特异性右臂突变前后与CD47的特异性结合情况:
NCI-H441细胞(人肺腺癌细胞)(购自北京北纳创联生物技术研究院),消化收集细胞并计数,离心并用PBS+2%FBS重悬细胞至3×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入各自12个稀释度的Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2、Hu5F9-G4、Ofatumumab(433.2nM、216.6nM,然后4倍梯度稀释,共12个浓度),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(Accuri C6,BD)检测荧光值。
表8流式细胞术检测双抗特异性右臂突变前后与CD47的特异性结合情况
例如,如图10和表8所示,除抗CD20抗体Ofatumumab不能结合CD47外,抗CD47抗体Hu5F9-G4、Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1m-Fc2均能与CD47结合,且Ofa-Fc1-D1m-Fc2(EC50=0.529nM)与NCI-H441细胞的结合亲和力高于Ofa-Fc1-D1-Fc2(EC50=8.68nM),其中D1m是SIRPα在D1区域的高亲和突变体(即CN106519036A的Seq ID No:10)。
重组蛋白早期体外免疫安全性评价实验
(1)制备人效应细胞悬液:
取生长良好的稳定高表达CD16a的人NK92MI-CD16a效应细胞(购于华博生物),离心(201g,5min)弃上清,重新悬浮在5ml MEM(无酚红)基础培养基(购自Gibco,51200-038)中,计数后,用MEM(无酚红)基础培养基调整靶细胞密度至2.4×106个细胞/ml,用作人效应细胞悬液。
(2)效应细胞与抗体的孵育:
将MEM(无酚红)基础培养基50μl/孔分液至96孔黑色底透培养板的对应孔中,加入稀释好的Ofa-Fc1-D1-Fc2或Ofa-Fc1-D1m-Fc2双特异性抗体稀释梯度各25μl/孔,并设置复孔。加入(1)中制备的人效应细胞悬液25μl(60000个细胞/孔)。混匀后Ofa-Fc1-D1-Fc2或Ofa-Fc1-D1m-Fc2双特异性抗体达到最终浓度梯度(433.2nM起始,4倍梯度稀释,共十个浓度),然后在37℃反应5.5小时,加入裂解缓冲液(来源于Promega的试剂盒,G7891)至对照组孵育0.5h。
(3)检测ADCC活性:
孵育完成后,将培养板置于安全柜中,开盖自然冷却至室温约15min。将室温平衡30min的LDH底物反应液(来源于Promega的试剂盒,G7891)加入培养板,每孔100μl,轻轻混匀后室温孵育15min后,立刻加入终止液(来源于Promega的试剂盒,G7891)50μl/孔,混匀后,于酶标仪上读取荧光值。
试验结果显示,由于NK细胞上有CD47抗原的表达,因此具有ADCC活性的靶向CD47的重组蛋白和/或抗体会导致NK细胞的相互杀伤作用。因此,相比于含SIRPα胞外截短体高亲和突变体的Ofa-Fc1-D1m-Fc2或Ofa-Fc1-D1m-D2-Fc2或Anti-EGFR-Fc1-D1m-Fc2或Anti-EGFR-Fc1-D1m-D2-Fc2或Anti-Her2(T)-Fc1-D1m-Fc2或Anti-Her2(T)-Fc1-D1m-D2-Fc2或Anti-Her2(P)-Fc1-D1m-Fc2或Anti-Her2(P)-Fc1-D1m-D2-Fc2或Anti-PD-L1(Ate)-Fc1-D1m-Fc2或Anti-PD-L1(Ate)-Fc1-D1m-D2-Fc2或Anti-PD-L1(13G4)-Fc1-D1m-Fc2或Anti-PD-L1(13G4)-Fc1-D1m-D2-Fc2或Anti-PD-L1(12A4)-Fc1-D1m-Fc2或Anti-PD-L1(12A4)-Fc1-D1m-D2-Fc2,Ofa-Fc1-D1-Fc2或Ofa-Fc1-D1-D2-Fc2或Ofa-Fc1-D1-D2-D3-Fc2或Obi-Fc1-D1-Fc2或Obi-Fc1-D1-D2-Fc2或Obi-Fc1-D1-D2-D3-Fc2或Anti-EGFR-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-D2-Fc2或Anti-EGFR-Fc1-D1-D2-D3-Fc2或Anti-Her2(P)-Fc1-D1-Fc2或Anti-Her2(P)-Fc1-D1-D2-Fc2或Anti-Her2(P)-Fc1-D1-D2-D3-Fc2或Anti-Her2(T)-Fc1-D1-Fc2或Anti-Her2(T)-Fc1-D1-D2-Fc2或Anti-Her2(T)-Fc1-D1-D2-D3-Fc2或Anti-EGFR-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-D2-Fc2或Anti-EGFR-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(Ate)-Fc1-D1-Fc2或Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2或Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(13G4)-Fc1-D1-Fc2或Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2或Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(12A4)-Fc1-D1-Fc2或Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2或Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2因其与CD47抗原的弱亲和作用,明显降低NK细胞所导致的毒副作用,毒副作用至少降低1000倍。
例如,如图11所示,当抗体/重组蛋白浓度达到10-1nM时,Ofa-Fc1-D1m-Fc2已开始出现细胞裂解,当抗体/重组蛋白浓度达到103nM时,Ofa-Fc1-D1m-Fc2其细胞裂解率已达到15.25%,而Ofa-Fc1-D1-Fc2在103nM时尚未观察到细胞裂解的情况。
如本领域技术人员所知,以上试验结果提示,本发明所述的具有ADCC活性且低亲和靶向CD47抗原的重组蛋白具有更高的免疫安全性。
如本领域技术人员所知,以上试验结果提示,使用本发明所述的优化后的ADCC活性检测的方法(即早期体外免疫安全性评价实验),可用于评价靶向CD47且具有ADCC活性的重组蛋白(含单价或多价)或抗体(含单价或多价)的早期免疫安全性,该评价方法简单快捷且不受血源的限制。
实施例5重组蛋白体内抑制肿瘤细胞生长实验
重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2体内抑制肿瘤细胞生长实验,以下方法以Ofa-Fc1-D1-Fc2为例,适用于右臂含SIRPα胞外截短体的重组蛋白的检测。
以人B细胞淋巴瘤Raji细胞皮下接种雄性NSG小鼠(购自艾德摩),在接种肿瘤细胞后待肿瘤组织达到80mm3-150mm3,分成以下2组(每组6只小鼠,每组按照标示量分别腹腔注射):1)溶媒对照组(Tris-柠檬酸盐,pH 6.5);2)Ofa-Fc1-D1-Fc2组(150μg/只);每周给药2次,共给药2周。分别在给药前(0天)、给药后第3天、第5天、第7天、第10天、第12天、第14天观察肿瘤生长情况,测量肿瘤大小以评价Ofa-Fc1-D1-Fc2的抑瘤作用。
试验结果显示,在Raji淋巴瘤皮下移植的NSG小鼠模型上,重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2通过阻断CD47-SIRPα信号通路,激活了巨噬细胞的靶向吞噬作用和/或巨噬细胞介导的抗体依赖的细胞吞噬作用(Antibody dependent cellular phagocytosis,ADCP),从而显示出显著的肿瘤抑制效果。
例如,如图12所示,横坐标为Raji淋巴瘤皮下移植的NSG小鼠接受药物处理的时间(天),纵坐标为肿瘤体积(mm3)。图12表明经过一段时间的药物Ofa-Fc1-D1-Fc2治疗后,较之于溶媒对照组,Ofa-Fc1-D1-Fc2组显示出了显著的肿瘤抑制趋势,其中,第14天时Ofa-Fc1-D1-Fc2组的抑瘤率可达到63.14%。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白在肿瘤细胞皮下移植的NSG小鼠体内可获得显著抑瘤功效。
实施例6重组蛋白体内早期免疫安全性评价实验
重组蛋白Ofa-Fc1-D1m-Fc2、Ofa-Fc1-D1m-D2-Fc2、Anti-EGFR-Fc1-D1m-Fc2、Anti-EGFR-Fc1-D1m-D2-Fc2、Anti-Her2(T)-Fc1-D1m-Fc2、Anti-Her2(T)-Fc1-D1m-D2-Fc2、Anti-Her2(P)-Fc1-D1m-Fc2、Anti-Her2(P)-Fc1-D1m-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-Fc2、Anti-PD-L1(Ate)-Fc1-D1m-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-Fc2、Anti-PD-L1(13G4)-Fc1-D1m-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-Fc2、Anti-PD-L1(12A4)-Fc1-D1m-D2-Fc2体内早期免疫安全性评价,以下方法以Ofa-Fc1-D1m-Fc2为例,适用于右臂含SIRPα胞外截短体高亲和突变体的重组蛋白的检测。
重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2体内早期免疫安全性评价,以下方法以Ofa-Fc1-D1-Fc2为例,适用于右臂含SIRPα胞外截短体的重组蛋白的检测。
因B细胞上高表达CD20抗原,本实验通过B细胞含量测定,代表肿瘤细胞杀伤情况。测定靶向其他肿瘤抗原和CD47抗原的重组蛋白体内早期免疫安全性评价时,需在本实施例所述实验小鼠皮下移植对应的肿瘤细胞。
肿瘤特异性靶向作用:
选择人CD34+HSC移植NSG(Hu-NSG)雌性小鼠(购自艾德摩),分成以下3组(每组3只小鼠,每组按照标示量分别静脉注射):1)0.9%生理盐水对照组;2)Hu5F9-G4(6.7μg/只)组;3)Ofa-Fc1-D1-Fc2(5μg/只)组,给药1次,给药后96h,小鼠尾静脉采血80μl于含肝素钠的抗凝管中,采用新鲜配制的裂红液裂解红细胞(其中裂红液为裂解液(BDPharm LyseTM,货号:555899):双蒸水等体积混合配制),剩余细胞用PBS+2%FBS清洗重悬,荧光抗体(PEanti-human CD45(货号:304039)/FITC anti-human CD19(货号:302206)/APC anti-humanCD3(货号:300312),均购自BioLegend)与细胞共孵育30min,PBS+2%FBS清洗重悬后,通过流式细胞仪(AccuriTM C6,BD)检测。
试验结果显示,在Hu-NSG小鼠模型使用同剂量的Ofa-Fc1-D1-Fc2和抗CD47抗体Hu5F9-G4,用药96小时后,Ofa-Fc1-D1-Fc2优先清除表达CD20抗原的B细胞(即靶细胞),而抗CD47抗体Hu5F9-G4因其与CD47抗原的高亲和作用则优先清除CD47表达丰度较高的非靶细胞,如T细胞等。
例如,如图13所示,抗CD47抗体Hu5F9-G4(图13B)相比于0.9%生理盐水(图13A),CD47表达丰度较高的非靶细胞(如T细胞)显著被清除(B细胞,即CD20抗原靶细胞,在所有被检测细胞中占比由用药前的40.9%上升至用药96小时后的73.5%);而重组蛋白Ofa-Fc1-D1-Fc2(图13C)相比于0.9%生理盐水(图13A),B细胞(即CD20抗原靶细胞)被显著清除,即用药后96小时,Ofa-Fc1-D1-Fc2优先清除B细胞(B细胞在所有被检测细胞中占比由用药前的40.9%下降至用药96小时后的3.7%)。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白在同剂量条件下优先清除肿瘤靶向抗原所在细胞和/或肿瘤细胞。
低剂量下的免疫安全性:
选择人CD34+HSC移植NSG(Hu-NSG)雌性小鼠(购自艾德摩),分成以下2组(每组3只小鼠,每组按照标示量分别静脉注射):1)Ofa-Fc1-D1-Fc2(1μg/只)组;2)Ofa-Fc1-D1m-Fc2(1μg/只)组,给药1次,给药后72h,小鼠尾静脉采血80μl于含肝素钠的抗凝管中,采用新鲜配制的裂红液裂解红细胞(其中裂红液为裂解液(BD Pharm LyseTM,货号:555899):双蒸水等体积混合配制),剩余细胞用PBS+2%FBS清洗重悬,荧光抗体(PE anti-human CD45(货号:304039)/FITCanti-human CD19(货号:302206)/APC anti-human CD3(货号:300312),均购自BioLegend)与细胞共孵育30min,PBS+2%FBS清洗重悬后,Ofa-Fc1-D1-Fc2(1μg/只)组样品通过流式细胞仪(AccuriTM C6,BD)检测,Ofa-Fc1-D1m-Fc2(1μg/只)组样品通过流式细胞仪(NovoCyteTM3130,ACEA)检测。
试验结果显示,在Hu-NSG小鼠模型使用低剂量的重组蛋白,当Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2单次用药72小时后,表达左臂抗原的靶细胞(如肿瘤细胞)被显著清除,而不表达左臂抗原的细胞(如T细胞、其他免疫细胞)则未见明显影响;而含SIRPα胞外截短体高亲和突变体的Ofa-Fc1-D1m-D2-Fc2或Anti-EGFR-Fc1-D1m-Fc2或Anti-EGFR-Fc1-D1m-D2-Fc2或Anti-Her2(T)-Fc1-D1m-Fc2或Anti-Her2(T)-Fc1-D1m-D2-Fc2或Anti-Her2(P)-Fc1-D1m-Fc2或Anti-Her2(P)-Fc1-D1m-D2-Fc2或Anti-PD-L1(Ate)-Fc1-D1m-Fc2或Anti-PD-L1(Ate)-Fc1-D1m-D2-Fc2或Anti-PD-L1(13G4)-Fc1-D1m-Fc2或Anti-PD-L1(13G4)-Fc1-D1m-D2-Fc2或Anti-PD-L1(12A4)-Fc1-D1m-Fc2或Anti-PD-L1(12A4)-Fc1-D1m-D2-Fc2单次用药72小时后,尽管表达左臂抗原的靶细胞(如肿瘤细胞)被显著清除,但不表达左臂抗原的细胞(如T细胞、其他免疫细胞)亦均有明显程度的清除。
例如,如图14所示,Ofa-Fc1-D1-Fc2单次给药后72小时(图14C、图14D)相比于给药前(图14A、图14B),B细胞(CD20抗原靶细胞)被显著清除(几乎完全被清除),而不表达CD20抗原的细胞(如T细胞、其他免疫细胞)则未见明显影响。Ofa-Fc1-D1m-Fc2单次给药后72小时(图14G、图14H)相比于给药前(图14E、图14F),尽管B细胞(CD20抗原靶细胞)被显著清除,但不表达CD20抗原的细胞(如T细胞、其他免疫细胞)亦均有明显程度的清除。
如本领域技术人员所知,以上试验结果提示,右臂含SIRPα胞外截短体的重组蛋白,在同剂量条件下,比右臂含SIRPα胞外截短体高亲和突变体的重组蛋白具有更高肿瘤特异性靶向作用,表现出更高的免疫安全性。
高剂量下的免疫恢复作用:
选择人CD34+HSC移植NSG(Hu-NSG)雌性小鼠(购自艾德摩),分成以下2组(每组3只小鼠,每组按照标示量分别静脉注射):1)Hu5F9-G4(200μg/只)组;2)Ofa-Fc1-D1-Fc2(150μg/只)组,给药1次,给药后4天及给药后14天,小鼠尾静脉采血80μl于含肝素钠的抗凝管中,采用新鲜配制的裂红液裂解红细胞(其中裂红液为裂解液(BDPharm LyseTM,货号:555899):双蒸水等体积混合配制),剩余细胞用PBS+2%FBS清洗重悬,荧光抗体(PE anti-humanCD45(货号:304039)/FITC anti-human CD19(货号:302206)/APC anti-human CD3(货号:300312),均购自BioLegend)与细胞共孵育30min,PBS+2%FBS清洗重悬后,通过流式细胞仪(AccuriTM C6,BD)检测。
试验结果显示,在Hu-NSG小鼠模型使用高剂量的Ofa-Fc1-D1-Fc2或Ofa-Fc1-D1-D2-Fc2或Ofa-Fc1-D1-D2-D3-Fc2或Obi-Fc1-D1-Fc2或Obi-Fc1-D1-D2-Fc2或Obi-Fc1-D1-D2-D3-Fc2或Anti-EGFR-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-D2-Fc2或Anti-EGFR-Fc1-D1-D2-D3-Fc2或Anti-Her2(P)-Fc1-D1-Fc2或Anti-Her2(P)-Fc1-D1-D2-Fc2或Anti-Her2(P)-Fc1-D1-D2-D3-Fc2或Anti-Her2(T)-Fc1-D1-Fc2或Anti-Her2(T)-Fc1-D1-D2-Fc2或Anti-Her2(T)-Fc1-D1-D2-D3-Fc2或Anti-EGFR-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-D2-Fc2或Anti-EGFR-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(Ate)-Fc1-D1-Fc2或Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2或Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(13G4)-Fc1-D1-Fc2或Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2或Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(12A4)-Fc1-D1-Fc2或Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2或Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2和抗CD47抗体Hu5F9-G4,单次给药96小时后,Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2和抗CD47抗体Hu5F9-G4均可见B细胞(CD20抗原靶细胞)及非靶细胞(如T细胞及其他免疫细胞)被较大程度的清除现象。14天后,Ofa-Fc1-D1-Fc2或Ofa-Fc1-D1-D2-Fc2或Ofa-Fc1-D1-D2-D3-Fc2或Obi-Fc1-D1-Fc2或Obi-Fc1-D1-D2-Fc2或Obi-Fc1-D1-D2-D3-Fc2或Anti-EGFR-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-D2-Fc2或Anti-EGFR-Fc1-D1-D2-D3-Fc2或Anti-Her2(P)-Fc1-D1-Fc2或Anti-Her2(P)-Fc1-D1-D2-Fc2或Anti-Her2(P)-Fc1-D1-D2-D3-Fc2或Anti-Her2(T)-Fc1-D1-Fc2或Anti-Her2(T)-Fc1-D1-D2-Fc2或Anti-Her2(T)-Fc1-D1-D2-D3-Fc2或Anti-EGFR-Fc1-D1-Fc2或Anti-EGFR-Fc1-D1-D2-Fc2或Anti-EGFR-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(Ate)-Fc1-D1-Fc2或Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2或Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(13G4)-Fc1-D1-Fc2或Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2或Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2或Anti-PD-L1(12A4)-Fc1-D1-Fc2或Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2或Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2组的小鼠,其B细胞(CD20抗原靶细胞)仍然处于被清除的状态,而表达CD47的其他非靶细胞(如T细胞)则显著得到恢复;而抗CD47抗体Hu5F9-G4组的小鼠,无论是B细胞(CD20抗原靶细胞)还是表达CD47的非靶细胞均未恢复。
例如,如图15所示,高剂量的抗CD47抗体Hu5F9-G4(图15A)和Ofa-Fc1-D1-Fc2(图15B)虽在给药后96小时均出现了B细胞(CD20抗原靶细胞)和表达CD47的非靶细胞(如T细胞)的大量清除,但14天后,Ofa-Fc1-D1-Fc2(图15D)组小鼠虽B细胞(CD20抗原靶细胞)仍处于被清除的状态,但B细胞(CD20抗原靶细胞)以外表达CD47的非靶细胞(如T细胞)显著得到恢复,而抗CD47抗体Hu5F9-G4(图15C)组小鼠,其B细胞(CD20抗原靶细胞)、表达CD47的非靶细胞(如T细胞)均未有恢复迹象。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白,其表达CD47的非靶细胞(如T细胞等免疫细胞)在高剂量给药条件下具有可恢复性,该重组蛋白具有更高的免疫安全性。
如本领域技术人员所知,以上试验结果提示,使用本发明所述的体内早期免疫安全性评价方法,可用于评价靶向CD47重组蛋白(含单价或多价)或抗体(含单价或多价)的早期免疫安全性。
实施例7不同截短体对靶标亲和力结合活性的影响
重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2以Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-Fc2和Anti-Her2(P)-Fc1-D1-D2-Fc2为例,适用于左臂相同,右臂采用不同长度的SIRPα胞外截短体的重组蛋白。
流式细胞术检测与靶标Her2和CD47的双特异性结合情况:
SKBR-3细胞(人乳腺癌细胞)(购自上海中科院细胞库),收集生长良好的细胞并计数,离心并用PBS+2%FBS重悬细胞至2×106个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号:3799,Corning),静置至少15分钟;离心吸出上清并分别加入各自11个稀释度的Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-Fc2和Anti-Her2(P)-Fc1-D1-D2-Fc2(433.2nM起始、4倍梯度连续稀释,共11个浓度),4℃孵育1小时;PBS+2%FBS清洗后,加入山羊抗人IgG Fc-FITC(F9512-2ML,Sigma)4℃孵育1小时;用PBS+2%FBS清洗重悬后,通过流式细胞仪(Accuri C6,BD)检测荧光值。
由于Trastuzumab和Pertuzumab分别作用于Her2抗原的不同表位,且2个抗原表位与细胞膜间距离存在较大差异,因此,重组蛋白Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2均能与SKBR-3细胞特异性结合,但结合亲和力以及所能达到的最大平均荧光强度各不相同。
试验结果显示,由于Trastuzumab与Her2抗原表位(Pedersen M W,etal.Targeting Three Distinct HER2Domains with a Recombinant Antibody MixtureOvercomes Trastuzumab Resistance.Molecular Cancer Therapeutics,2015,14(3):669-680)距离细胞膜表面更近,因此Anti-Her2(T)-Fc1-D1-Fc2与SKBR-3细胞亲和力优于Anti-Her2(T)-Fc1-D1-D2-Fc2。Pertuzumab与Her2抗原表位(Her2胞外第II结构域)距离细胞膜表面较远,因此Anti-Her2(P)-Fc1-D1-Fc2较Anti-Her2(P)-Fc1-D1-D2-Fc2与SKBR-3细胞亲和力相当。
以上试验数据证明,右臂不同长度截短体组成的重组蛋白会影响重组蛋白与靶细胞的亲和力。对于结合近膜端表位的左臂,当右臂选择较短的SIRPα截短体时,能大大提高重组蛋白结合双靶点的能力。但对于结合远膜端表位的左臂,右臂选择较短的SIRPα截短体就失去了优势,左臂抗原靶点距离细胞膜越远,右臂的SIRPα截短体就应该越长才能达到最优匹配。
例如,如图16所示,Anti-Her2(T)-Fc1-D1-Fc2(EC50=2.04nM)结合SKBR-3细胞的能力明显优于Anti-Her2(T)-Fc1-D1-D2-Fc2(EC50=25.95nM)(图16A)。而Anti-Her2(P)-Fc1-D1-Fc2结合SKBR-3细胞的能力(EC50=15.22nM)则与Anti-Her2(P)-Fc1-D1-D2-Fc2(EC50=11.03nM)相当(图16B)。
如本领域技术人员所知,以上试验结果提示,根据靶抗原空间表位与靶细胞膜表面的距离,右臂适配性的选择合适长度的人SIRPα胞外截短体,可有效提高重组蛋白与靶细胞的结合能力。
实施例8重组蛋白急性毒性试验
重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2的急性毒性试验,以下方法以Ofa-Fc1-D1-Fc2为例,适用于右臂含SIRPα胞外截短体的重组蛋白的检测。
取适量的Ofa-Fc1-D1-Fc2溶液(4.02mg/mL),用注射用Ofa-Fc1-D1-Fc2缓冲液(Tris-柠檬酸盐,pH 6.5)将其分别稀释至0.25和2.5mg/mL,分别用于实验动物第1组和第2组的给药。
4只健康雌性食蟹猴,购于广西桂东灵长类开发实验有限公司,年龄为4岁,由广西壮族自治区科学技术厅许可上述实验动物的生产,实验动物生产许可证编号为SCXK桂2016-0001。给药前进行一次详细临床观察和称重,均未见异常。体重范围在给药当天为2.47-2.85kg。
表9实验设计
*通过静脉注射给药
将4只雌性食蟹猴分为两组,每组两只,通过静脉注射给药,给药剂量为0.5mg/kg、5mg/kg,给药体积为2mL/kg。实验设计见上表9。给药当天给药一次,给药结束后连续观察28天。食蟹猴饲养于不锈钢移动笼中,每笼1只。每天提供大约各12小时的明暗交替照明。实验猴配合饲料购于北京科澳协力饲料有限公司,试验期间动物自由采食,特定禁食除外。该批饲料由上海谱尼测试技术有限公司(PONY)对其中特定的微生物、重金属和农药残留进行检测。试验期间所有动物通过水瓶自由饮水,所用饮用水为反渗透系统过滤除菌的纯化水,由计量员对饮用水的pH、硬度、重金属和微生物进行检测。
试验期间所有试验动物每天进行两次(上、下午各一次)笼边观察,观察内容包括但不限于发病、损害、死亡和供食供水情况。所有受试动物试验前进行1次详细临床观察。试验过程中所有实验动物给药后每天至少1次进行详细临床观察。观察内容包括但不限于发病、死亡率、损害和供食供水情况,皮肤,毛,眼,耳,鼻,口腔,胸部,腹部,外生殖器,四肢,呼吸及循环系统,自主效应(如流涎),神经系统(如震颤,抽搐,应激反应以及反常行为)。分别于D-1(给药前),D1,D4,D8,D11,D15,D18,D22,D25及解剖前,测定动物体重。分别于D2,D4,D8,D11,D15,D18,D22,D25测定动物24h内(24h±1h)耗食量。D-1,D2,D14,D28进行心电图测定,采用标准II导联(8个导联)进行记录,记录速度50mm/秒。
分别在给药前(D-1)、给药后D2、D7、D14和D28进行临床病理学样本采集及血液学、凝血、血液生化和淋巴细胞分型检测;于给药前、给药后第28天采集尿液样本并进行尿液分析。
样本采集前所有动物禁食过夜(至少10小时)但不禁水。股静脉采集血样(4.5-6mL),其中大约1.8mL全血于含有枸橼酸钠的抗凝管中用于凝血分析;大约1mL全血置于含有K3-EDTA的抗凝管用于血液学分析;大约2mL全血置于分离胶采血管(无抗凝剂)用于血液生化学分析,根据标准作业程序要求离心分离血清。同时,对给药前D-1、给药后D2分离后的血清样本采用流式细胞仪进行T/B细胞分型检测。
在D29对动物进行安乐死,采集并保存心脏、肝脏、脾脏、肺和肾组织,并对肝脏、肺(含主支气管)、肾脏、脾脏、心脏、肾上腺、垂体、甲状腺和甲状旁腺、胸腺、卵巢、子宫(包括子宫颈)、脑等组织进行称重。
结果显示,单次静脉注射给予Ofa-Fc1-D1-Fc2,给药剂量分别为0.5、5mg/kg时,给药后连续观察28天,动物未见明显药物作用相关异常,且此期间动物耗食量、体重均在正常范围内波动;给药后与给药前相比,动物凝血、尿液及心电图相关检测数据与给药前相比无显著性变化;动物进行解剖后,所有脏器大体观察均在正常范围内,动物脏器重量、脏体系数、脏脑比也在正常范围内。
给药后D2,低、高剂量组动物淋巴细胞计数及淋巴细胞百分比均出现明显下降,D7之后恢复至正常水平,此变化可能与药物作用相关。
重组蛋白Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2在同等剂量下与Ofa-Fc1-D1-Fc2上述结果情况类似。
表10重组蛋白对食蟹猴红细胞数量(1012个细胞/L)的影响
如图18A及表10所示,0.5mg/kg和5mg/kg两种剂量的Ofa-Fc1-D1-Fc2均未对食蟹猴红细胞数量产生影响;如图18B及表10所示,0.5mg/kg和5mg/kg两种剂量的Ofa-Fc1-D1-Fc2,均未对食蟹猴血红蛋白产生影响。
表11重组蛋白对食蟹猴血红蛋白(g/L)的影响
重组蛋白Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2在同等剂量下对食蟹猴红细胞数量以及血红蛋白的影响与Ofa-Fc1-D1-Fc2上述结果情况类似。
如图19所示,根据T/B细胞分型检测结果显示,0.5mg/kg和5mg/kg两种剂量的Ofa-Fc1-D1-Fc2,可导致动物的B细胞(CD20抗原靶细胞)被显著清除。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白,单次给药后动物未见明显药物作用相关异常,且此期间动物耗食量、体重均在正常范围内波动;给药后与给药前相比,动物凝血、尿液及心电图相关检测数据与给药前相比无显著性变化;动物进行解剖后,所有脏器大体观察均在正常范围内,动物脏器重量、脏体系数、脏脑比也在正常范围内。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白,单次给药后,未对动物红细胞数量及血红蛋白数量产生影响。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白在同等剂量条件下优先清除肿瘤靶向抗原所在细胞和/或肿瘤细胞。
实施例9重组蛋白体内抑制肿瘤生长实验
重组蛋白Ofa-Fc1-D1-Fc2、Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2体内抑制肿瘤生长实验,以下方法以Ofa-Fc1-D1-Fc2为例,适用于右臂含SIRPα胞外截短体的重组蛋白的检测。
Ofa-Fc1-D1-Fc2:无色澄清液体,浓度1.14-4.02mg/ml,-80℃分装保存;
(利妥昔单抗注射液):无色澄清液体,规格100mg/10ml,批号H0205,2-8℃避光保存。制剂缓冲液(Tris-柠檬酸盐,pH 6.5):无色澄清液体,2-8℃保存。配制方法:Ofa-Fc1-D1-Fc2、
均用制剂缓冲液稀释;制剂缓冲液作为溶剂直接给药。
细胞:CD20阳性人B细胞淋巴瘤Daudi细胞购自中国科学院细胞库。培养条件为RPMI 1640培养基中加10%胎牛血清以及青、链霉素,于37℃、含5%CO2空气的培养箱中培养。一周二次传代,当细胞呈指数生长期时,收集细胞,计数,接种。
实验动物:雌性NOD-SCID小鼠,6-7周,购自上海灵畅生物科技有限公司。生产许可证号:SCXK(沪)2013-0018;动物合格证号2013001829463、2013001827545。饲养环境:SPF级。本实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况,例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动、体重变化、外观体征等。
每只小鼠皮下接种1.5×107Daudi细胞,接种后第18天当平均肿瘤体积达到100-150mm3时,根据肿瘤体积分组并给药(D0)。小鼠静脉注射(IV)药物;溶剂组注射相同体积的溶剂,注射体积0.1mL/10g体重;给药剂量和给药方案参见表12。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为:
肿瘤体积(V)计算公式为:
V=1/2×a×b2
其中a、b分别表示长、宽。
T/C(%)=(T-T0)/(C-C0)×100
其中T、C为实验结束时的肿瘤体积;T0、C0为实验开始时的肿瘤体积;T为给药组,C为对照组。
抑瘤率(TGI)(%)=100-T/C(%)。
当肿瘤出现消退时,抑瘤率(TGI)(%)=100-(T-T0)/T0×100
如果肿瘤比起始体积缩小,即T<T0或C<C0时,即定义为肿瘤部分消退(PR);如果肿瘤完全消失,即定义为肿瘤完全消退(CR)。
实验结束后,或当动物的肿瘤体积达到1500mm3的安乐死终点时,二氧化碳麻醉处死动物,随后解剖取瘤并称重拍照。
两组肿瘤体积或肿瘤重量之间的比较采用双尾Student’s t检验,P<0.05定义为有统计学显著性差异。
Ofa-Fc1-D1-Fc2(5mg/kg,IV,每周2次,共5次)明显抑制Daudi皮下移植瘤的生长,抑瘤率为80.8%,1/6小鼠肿瘤部分消退;
(7mg/kg,IV,每周2次,共5次)对Daudi皮下移植瘤的抑瘤率为24.5%。荷瘤小鼠对以上药物总体上能较好耐受(表12,图20、21、22)。
表12 Ofa-Fc1-D1-Fc2、
对人B细胞淋巴瘤Daudi细胞皮下移植瘤的疗效
注:随机分组,第一次给药时间为D0;IV:静脉注射。
可见,Ofa-Fc1-D1-Fc2和
对CD20阳性人B细胞淋巴瘤Daudi细胞皮下移植瘤生长均有不同程度的抑制作用,其中Ofa-Fc1-D1-Fc2显著优于
荷瘤小鼠对以上药物总体上能较好耐受。
重组蛋白Ofa-Fc1-D1-D2-Fc2、Ofa-Fc1-D1-D2-D3-Fc2、Obi-Fc1-D1-Fc2、Obi-Fc1-D1-D2-Fc2、Obi-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-Her2(P)-Fc1-D1-Fc2、Anti-Her2(P)-Fc1-D1-D2-Fc2、Anti-Her2(P)-Fc1-D1-D2-D3-Fc2、Anti-Her2(T)-Fc1-D1-Fc2、Anti-Her2(T)-Fc1-D1-D2-Fc2、Anti-Her2(T)-Fc1-D1-D2-D3-Fc2、Anti-EGFR-Fc1-D1-Fc2、Anti-EGFR-Fc1-D1-D2-Fc2、Anti-EGFR-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(Ate)-Fc1-D1-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-Fc2、Anti-PD-L1(Ate)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(13G4)-Fc1-D1-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-Fc2、Anti-PD-L1(13G4)-Fc1-D1-D2-D3-Fc2、Anti-PD-L1(12A4)-Fc1-D1-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-Fc2、Anti-PD-L1(12A4)-Fc1-D1-D2-D3-Fc2在同等剂量下对NOD-SCID小鼠体内移植瘤的抑瘤效果及小鼠对重组蛋白的耐受程度与Ofa-Fc1-D1-Fc2上述结果情况类似。
如本领域技术人员所知,以上试验结果提示,本发明所述的可以与肿瘤靶向抗原和CD47抗原同步结合的重组蛋白在同等剂量条件下相较于靶向肿瘤的抗体,在小鼠体内有意想不到的显著抑瘤效果。
本实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况,例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动、体重变化、外观体征等。
本文提供的任何和所有实施例或示例性语言(例如,“诸如”)的使用仅旨在更好地说明本发明,而不对本发明的范围构成限制,除非另有要求。说明书中的语言不应被解释为指示任何未要求保护的元件对于实施本发明是必要的。
本说明书中引用的所有出版物和专利申请通过引用并入本文,如同每个单独的出版物或专利申请被具体地和单独地指明通过引用并入。此外,本文所述的任何理论、机制、证明或发现旨在进一步增强对本发明的理解,并且不意图以任何方式将本发明限制到这样的理论、机制、证明或发现。尽管已经在附图和前面的描述中详细地示出和描述了本发明,但是本发明应当被认为是说明性的而不是限制性的。
序列表
<110> 上海津曼特生物科技有限公司
<120> 双特异性重组蛋白及其应用
<130> IDC170239
<150> CN201710317926.7
<151> 2017-05-08
<150> CN201711269620.5
<151> 2017-12-05
<160> 44
<170> PatentIn version 3.5
<210> 1
<211> 471
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofa重链氨基酸序列
<400> 1
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys
85 90 95
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu
100 105 110
Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met
115 120 125
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
130 135 140
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
145 150 155 160
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
180 185 190
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
210 215 220
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
225 230 235 240
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 2
<211> 234
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofa轻链氨基酸序列
<400> 2
Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Asp Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
20 25 30
Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
35 40 45
Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
100 105 110
Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 3
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Obi重链氨基酸序列
<400> 3
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
Ser Tyr Ser Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
50 55 60
Glu Trp Met Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn
65 70 75 80
Gly Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 4
<211> 239
<212> PRT
<213> Artificial Sequence
<220>
<223> Obi/Obi-Fc1轻链氨基酸序列
<400> 4
Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Asp Ala Arg Cys Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
20 25 30
Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser
35 40 45
Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys
50 55 60
Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val
65 70 75 80
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
85 90 95
Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
100 105 110
Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys
115 120 125
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
130 135 140
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
145 150 155 160
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
165 170 175
Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
180 185 190
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
195 200 205
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
210 215 220
Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 5
<211> 461
<212> PRT
<213> Artificial Sequence
<220>
<223> Hu5F9-G4重链氨基酸序列
<400> 5
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
20 25 30
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
35 40 45
Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
50 55 60
Met Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys
65 70 75 80
Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala
85 90 95
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
100 105 110
Cys Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr
115 120 125
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135 140
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
145 150 155 160
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
165 170 175
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
180 185 190
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
195 200 205
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
210 215 220
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
225 230 235 240
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
275 280 285
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
290 295 300
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
305 310 315 320
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
325 330 335
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
355 360 365
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
385 390 395 400
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
405 410 415
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
420 425 430
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
450 455 460
<210> 6
<211> 236
<212> PRT
<213> Artificial Sequence
<220>
<223> Hu5F9-G4轻链氨基酸序列
<400> 6
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro
20 25 30
Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr
35 40 45
Ser Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln
50 55 60
Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
65 70 75 80
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
85 90 95
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln
100 105 110
Gly Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
115 120 125
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
130 135 140
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
165 170 175
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
180 185 190
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
210 215 220
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 7
<211> 466
<212> PRT
<213> Artificial Sequence
<220>
<223> JMT101重链氨基酸序列
<400> 7
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
20 25 30
Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn
35 40 45
Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
50 55 60
Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe
65 70 75 80
Thr Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
85 90 95
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
100 105 110
Ala Arg Ala Leu Asp Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln
115 120 125
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
130 135 140
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
145 150 155 160
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
165 170 175
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
180 185 190
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
195 200 205
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
210 215 220
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
225 230 235 240
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
245 250 255
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
260 265 270
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
275 280 285
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
290 295 300
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
305 310 315 320
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
325 330 335
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
340 345 350
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
355 360 365
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
370 375 380
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
385 390 395 400
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
405 410 415
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
420 425 430
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
435 440 445
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
450 455 460
Gly Lys
465
<210> 8
<211> 231
<212> PRT
<213> Artificial Sequence
<220>
<223> JMT101/Anti-EGFR-Fc1轻链氨基酸序列
<400> 8
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro
20 25 30
Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr
35 40 45
Asn Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu
50 55 60
Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser
65 70 75 80
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu
85 90 95
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Glu Trp Pro
100 105 110
Thr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
115 120 125
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
130 135 140
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
145 150 155 160
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
165 170 175
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
180 185 190
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
195 200 205
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
210 215 220
Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 9
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Trastuzumab重链氨基酸序列
<400> 9
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
20 25 30
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
35 40 45
Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
50 55 60
Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
65 70 75 80
Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
85 90 95
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
100 105 110
Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
210 215 220
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 10
<211> 231
<212> PRT
<213> Artificial Sequence
<220>
<223> Trastuzumab轻链氨基酸序列
<400> 10
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
20 25 30
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr
35 40 45
Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
50 55 60
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
65 70 75 80
Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
85 90 95
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro
100 105 110
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
115 120 125
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
130 135 140
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
145 150 155 160
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
165 170 175
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
180 185 190
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
195 200 205
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
210 215 220
Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 11
<211> 359
<212> PRT
<213> Artificial Sequence
<220>
<223> SIRPα D1-Fc氨基酸序列
<400> 11
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
130 135 140
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
145 150 155 160
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
165 170 175
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
180 185 190
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
195 200 205
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
210 215 220
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
225 230 235 240
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
245 250 255
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
260 265 270
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
275 280 285
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
290 295 300
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
305 310 315 320
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
325 330 335
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
340 345 350
Leu Ser Leu Ser Pro Gly Lys
355
<210> 12
<211> 467
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab重链氨基酸序列
<400> 12
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
85 90 95
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
210 215 220
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
225 230 235 240
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr
305 310 315 320
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
325 330 335
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
340 345 350
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
370 375 380
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
450 455 460
Pro Gly Lys
465
<210> 13
<211> 233
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab/Anti-PD-L1(Ate)-Fc1轻链氨基酸序列
<400> 13
Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly
1 5 10 15
Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
20 25 30
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val
35 40 45
Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
50 55 60
Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
65 70 75 80
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
85 90 95
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr
100 105 110
His Pro Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
115 120 125
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
130 135 140
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
145 150 155 160
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
165 170 175
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
180 185 190
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
195 200 205
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
210 215 220
Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 14
<211> 1092
<212> DNA
<213> Artificial Sequence
<220>
<223> D1-Fc2 DNA序列
<400> 14
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60
gaggagctgc aggtcatcca gcccgataag agcgtgtccg tggccgcagg agaatctgcc 120
atcctgcatt gcaccgtgac ctctctgatc cccgtgggcc caatccagtg gttcagagga 180
gccggaccag ctagagagct gatctacaac cagaaggagg gccacttccc cagagtgaca 240
accgtgtccg agtctaccaa gcgggagaac atggacttct ccatctccat ctccgccatc 300
acaccagccg acgccggcac ctactattgc gtgaagttcc ggaagggctc cccagatacc 360
gagtttaaga gcggcgccgg aacagagctg agcgtgcggg ctaagcctga caagacccac 420
acctgtcccc cttgtcctgc ccctgaactg ctgggcggac cttccgtgtt cctgttcccc 480
ccaaagccca aggacaccct gatgatctcc cggacccccg aagtgacctg cgtggtggtg 540
gatgtgtccc acgaggaccc tgaagtgaag ttcaattggt acgtggacgg cgtggaagtg 600
cacaacgcca agaccaagcc tagagaggaa cagtacaact ccacctaccg ggtggtgtcc 660
gtgctgaccg tgctgcacca ggattggctg aacggcaaag agtacaagtg caaggtgtcc 720
aacaaggccc tgcctgcccc catcgaaaag accatctcca aggccaaggg ccagccccgg 780
gaaccccagg tgtacacact gccccctagc agggacgagc tgaccaagaa ccaggtgtcc 840
ctgtggtgtc tcgtgaaagg cttctacccc tccgacattg ccgtggaatg ggagtccaac 900
ggccagcctg agaacaacta caagaccacc ccccctgtgc tggactccga cggctcattc 960
ttcctgtaca gcaagctgac agtggacaag tcccggtggc agcagggcaa cgtgttctcc 1020
tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gtccctgagc 1080
cccggcaaat ga 1092
<210> 15
<211> 1389
<212> DNA
<213> Artificial Sequence
<220>
<223> D1-D2-Fc2 DNA序列
<400> 15
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60
gaggagctgc aggtcatcca gcccgataag agcgtgtccg tggccgcagg agaatctgcc 120
atcctgcatt gcaccgtgac ctctctgatc cccgtgggcc caatccagtg gttcagagga 180
gccggaccag ctagagagct gatctacaac cagaaggagg gccacttccc cagagtgaca 240
accgtgtccg agtctaccaa gcgggagaac atggacttct ccatctccat ctccgccatc 300
acaccagccg acgccggcac ctactattgc gtgaagttcc ggaagggctc cccagatacc 360
gagtttaaga gcggcgccgg aacagagctg agcgtgcggg ctaagccttc tgctccagtg 420
gtgtcaggac cagcagctag agctacccct cagcacaccg tgtccttcac ctgcgagtct 480
cacggcttct cccctagaga catcaccctc aagtggttca agaacggcaa cgagctgtcc 540
gacttccaga ccaacgtgga tccagtgggc gagagcgtgt cttactccat ccactccacc 600
gccaaggtgg tgctgacaag ggaggacgtg cactcccagg tcatttgcga ggtggcacac 660
gtgacattgc agggcgaccc cctgagggga accgccaact tgagtgacaa gacccacacc 720
tgtccccctt gtcctgcccc tgaactgctg ggcggacctt ccgtgttcct gttcccccca 780
aagcccaagg acaccctgat gatctcccgg acccccgaag tgacctgcgt ggtggtggat 840
gtgtcccacg aggaccctga agtgaagttc aattggtacg tggacggcgt ggaagtgcac 900
aacgccaaga ccaagcctag agaggaacag tacaactcca cctaccgggt ggtgtccgtg 960
ctgaccgtgc tgcaccagga ttggctgaac ggcaaagagt acaagtgcaa ggtgtccaac 1020
aaggccctgc ctgcccccat cgaaaagacc atctccaagg ccaagggcca gccccgggaa 1080
ccccaggtgt acacactgcc ccctagcagg gacgagctga ccaagaacca ggtgtccctg 1140
tggtgtctcg tgaaaggctt ctacccctcc gacattgccg tggaatggga gtccaacggc 1200
cagcctgaga acaactacaa gaccaccccc cctgtgctgg actccgacgg ctcattcttc 1260
ctgtacagca agctgacagt ggacaagtcc cggtggcagc agggcaacgt gttctcctgc 1320
tccgtgatgc acgaggccct gcacaaccac tacacccaga agtccctgtc cctgagcccc 1380
ggcaaatga 1389
<210> 16
<211> 471
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofa-Fc1重链氨基酸序列
<400> 16
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Asn Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys
85 90 95
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu
100 105 110
Tyr Tyr Cys Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met
115 120 125
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
130 135 140
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
145 150 155 160
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
165 170 175
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
180 185 190
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
195 200 205
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
210 215 220
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
225 230 235 240
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 17
<211> 234
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofa-Fc1 轻链氨基酸序列
<400> 17
Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Asp Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
20 25 30
Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
35 40 45
Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60
Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
100 105 110
Asn Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 18
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Obi-Fc1重链氨基酸序列
<400> 18
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Ser Asn Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr
65 70 75 80
Pro Phe Thr Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg Ala Leu Asp Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 19
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-EGFR-Fc1重链氨基酸序列
<400> 19
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
20 25 30
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Ser Asn Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr
65 70 75 80
Pro Phe Thr Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
85 90 95
Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg Ala Leu Asp Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 20
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-Her2(T)-Fc1重链氨基酸序列
<400> 20
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
85 90 95
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 21
<211> 234
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-Her2(T)-Fc1轻链氨基酸序列
<400> 21
Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
35 40 45
Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr
100 105 110
Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 22
<211> 468
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-Her2(P)-Fc1重链氨基酸序列
<400> 22
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Thr Asp Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn
65 70 75 80
Gln Arg Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys
465
<210> 23
<211> 234
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-Her2(P)-Fc1 轻链氨基酸序列
<400> 23
Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
35 40 45
Val Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
50 55 60
Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
100 105 110
Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
180 185 190
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
195 200 205
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
210 215 220
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 24
<211> 467
<212> PRT
<213> Artificial Sequence
<220>
<223> Anti-PD-L1(Ate)-Fc1重链氨基酸序列
<400> 24
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Asp Ser Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
85 90 95
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
165 170 175
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
180 185 190
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
195 200 205
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
210 215 220
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
225 230 235 240
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
245 250 255
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
290 295 300
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
305 310 315 320
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
325 330 335
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
340 345 350
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
370 375 380
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
420 425 430
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
435 440 445
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
450 455 460
Pro Gly Lys
465
<210> 25
<211> 1641
<212> DNA
<213> Artificial Sequence
<220>
<223> D1-D2-D3-Fc2 DNA序列
<400> 25
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60
gaggagctgc aggtcatcca gcccgataag agcgtgtccg tggccgcagg agaatctgcc 120
atcctgcatt gcaccgtgac ctctctgatc cccgtgggcc caatccagtg gttcagagga 180
gccggaccag ctagagagct gatctacaac cagaaggagg gccacttccc cagagtgaca 240
accgtgtccg agtctaccaa gcgggagaac atggacttct ccatctccat ctccgccatc 300
acaccagccg acgccggcac ctactattgc gtgaagttcc ggaagggctc cccagatacc 360
gagtttaaga gcggcgccgg aacagagctg agcgtgcggg ctaagccttc tgctccagtg 420
gtgtcaggac cagcagctag agctacccct cagcacaccg tgtccttcac ctgcgagtct 480
cacggcttct cccctagaga catcaccctc aagtggttca agaacggcaa cgagctgtcc 540
gacttccaga ccaacgtgga tccagtgggc gagagcgtgt cttactccat ccactccacc 600
gccaaggtgg tgctgacaag ggaggacgtg cactcccagg tcatttgcga ggtggcacac 660
gtgacattgc agggcgaccc cctgagaggc acagcaaact tgagcgagac aattagagtg 720
ccccccaccc tggaagttac acagcagccc gttagagccg agaaccaggt caacgtcacc 780
tgccaggtca gaaagtttta tccacagaga ctgcagctga cctggctcga gaacggaaac 840
gtgagcagaa cagagaccgc cagcaccgtg acagagaaca aggacgggac ctacaactgg 900
atgagttggc tgctggtgaa cgtcagcgcc cacagagacg acgtcaagct gacctgcgac 960
aagacccaca cctgtccccc ttgtcctgcc cctgaactgc tgggcggacc ttccgtgttc 1020
ctgttccccc caaagcccaa ggacaccctg atgatctccc ggacccccga agtgacctgc 1080
gtggtggtgg atgtgtccca cgaggaccct gaagtgaagt tcaattggta cgtggacggc 1140
gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacaactc cacctaccgg 1200
gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1260
aaggtgtcca acaaggccct gcctgccccc atcgaaaaga ccatctccaa ggccaagggc 1320
cagccccggg aaccccaggt gtacacactg ccccctagca gggacgagct gaccaagaac 1380
caggtgtccc tgtggtgtct cgtgaaaggc ttctacccct ccgacattgc cgtggaatgg 1440
gagtccaacg gccagcctga gaacaactac aagaccaccc cccctgtgct ggactccgac 1500
ggctcattct tcctgtacag caagctgaca gtggacaagt cccggtggca gcagggcaac 1560
gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1620
tccctgagcc ccggcaaatg a 1641
<210> 26
<211> 363
<212> PRT
<213> Artificial Sequence
<220>
<223> D1-Fc2 氨基酸序列
<400> 26
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Asp Lys Thr His Thr Cys Pro Pro
130 135 140
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
145 150 155 160
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
165 170 175
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
180 185 190
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
195 200 205
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
210 215 220
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
225 230 235 240
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
245 250 255
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
260 265 270
Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe
275 280 285
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
290 295 300
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
305 310 315 320
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
325 330 335
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
340 345 350
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 27
<211> 462
<212> PRT
<213> Artificial Sequence
<220>
<223> D1-D2-Fc2 氨基酸序列
<400> 27
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser
180 185 190
Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Asp Lys Thr His Thr
225 230 235 240
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
245 250 255
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
260 265 270
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
275 280 285
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
290 295 300
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
305 310 315 320
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
325 330 335
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
340 345 350
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
355 360 365
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
370 375 380
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
385 390 395 400
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
405 410 415
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
420 425 430
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
435 440 445
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
<210> 28
<211> 546
<212> PRT
<213> Artificial Sequence
<220>
<223> D1-D2-D3-Fc2 氨基酸序列
<400> 28
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser
35 40 45
Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro
130 135 140
Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser
180 185 190
Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val
225 230 235 240
Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln
245 250 255
Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln
260 265 270
Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser
275 280 285
Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu
290 295 300
Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Asp
305 310 315 320
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
325 330 335
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
340 345 350
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
355 360 365
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
370 375 380
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
385 390 395 400
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
405 410 415
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
420 425 430
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
435 440 445
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
450 455 460
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
465 470 475 480
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
485 490 495
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
500 505 510
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
515 520 525
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
530 535 540
Gly Lys
545
<210> 29
<211> 369
<212> PRT
<213> Artificial Sequence
<220>
<223> D1m-Fc2 氨基酸序列
<400> 29
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser
35 40 45
Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Val Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser Asp Lys
130 135 140
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
145 150 155 160
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
165 170 175
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
180 185 190
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
195 200 205
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
210 215 220
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
225 230 235 240
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
245 250 255
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
260 265 270
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
275 280 285
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
290 295 300
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
305 310 315 320
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
325 330 335
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
340 345 350
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
355 360 365
Lys
<210> 30
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> D1 氨基酸序列
<400> 30
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro
115
<210> 31
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> D1-D2 氨基酸序列
<400> 31
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
115 120 125
Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe
130 135 140
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
145 150 155 160
Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr
165 170 175
Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His
180 185 190
Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
195 200 205
Leu Arg Gly Thr Ala Asn Leu Ser
210 215
<210> 32
<211> 300
<212> PRT
<213> Artificial Sequence
<220>
<223> D1-D2-D3 氨基酸序列
<400> 32
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg
115 120 125
Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe
130 135 140
Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu
145 150 155 160
Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr
165 170 175
Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His
180 185 190
Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro
195 200 205
Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr
210 215 220
Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val
225 230 235 240
Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp
245 250 255
Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr
260 265 270
Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn
275 280 285
Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys
290 295 300
<210> 33
<211> 123
<212> PRT
<213> Artificial Sequence
<220>
<223> D1m 氨基酸序列
<400> 33
Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala
1 5 10 15
Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu
35 40 45
Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser
100 105 110
Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser
115 120
<210> 34
<211> 1416
<212> DNA
<213> Artificial Sequence
<220>
<223> Ofa-Fc1重链DNA序列
<400> 34
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60
gtgcagctgg tggaatctgg cggcggactg gtgcagcctg gcagatccct gagactgtct 120
tgtgccgcct ccggcttcac cttcaacgac tacgccatgc actgggtgcg acaggcccct 180
ggcaaaggcc tggaatgggt gtccaccatc agctggaact ccggctccat cggctacgcc 240
gactccgtga agggccggtt caccatctcc cgggacaacg ccaagaagtc cctgtacctg 300
cagatgaact ccctgcgggc cgaggacacc gccctgtact actgtgccaa ggacatccag 360
tacggcaact actactacgg catggacgtg tggggccagg gcaccacagt gaccgtgtca 420
tctgcttcta ccaagggccc ctccgtgttt cctctggccc cttccagcaa gtccacctct 480
ggcggaacag ccgctctggg ctgcctcgtg aaggactact tccccgagcc tgtgaccgtg 540
tcctggaact ctggcgctct gacatccggc gtgcacacct tccctgctgt gctgcagtct 600
agcggcctgt actccctgtc ctccgtcgtg accgtgcctt ccagctctct gggcacccag 660
acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggaa 720
cccaagtcct gcgacaagac ccacacctgt cccccttgtc ctgcccctga actgctgggc 780
ggaccttccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat ctcccggacc 840
cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 900
tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 960
aactccacct accgggtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 1020
aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga aaagaccatc 1080
tccaaggcca agggccagcc ccgggaaccc caggtgtaca cactgccccc tagcagggac 1140
gagctgacca agaaccaggt gtccctgagc tgtgcagtga aaggcttcta cccctccgac 1200
attgccgtgg aatgggagtc caacggccag cctgagaaca actacaagac caccccccct 1260
gtgctggact ccgacggctc attcttcctg gtgagcaagc tgacagtgga caagtcccgg 1320
tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1380
acccagaagt ccctgtccct gagccccggc aaatga 1416
<210> 35
<211> 705
<212> DNA
<213> Artificial Sequence
<220>
<223> Ofa-Fc1轻链DNA序列
<400> 35
atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60
gagatcgtgc tgacccagtc tcctgccacc ctgtctctga gccctggcga gagagctacc 120
ctgtcctgca gagcctccca gtccgtgtcc tcttacctgg cctggtatca gcagaagccc 180
ggccaggctc cccggctgct gatctacgat gcctccaata gagccaccgg catccctgcc 240
agattctccg gctctggctc tggcaccgac tttaccctga ccatctccag cctggaaccc 300
gaggacttcg ccgtgtacta ctgccagcag cggtccaact ggcccatcac ctttggccag 360
ggcacccggc tggaaatcaa gagaaccgtg gccgctccct ccgtgttcat cttcccacct 420
tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 540
gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 600
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 660
ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga 705
<210> 36
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Obi-Fc1重链DNA序列
<400> 36
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctcag 60
gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gctcctccgt gaaggtgtcc 120
tgcaaggctt ccggctacgc cttctcctac tcctggatca actgggtgcg acaggcccct 180
ggacagggcc tggaatggat gggcagaatc ttccctggcg acggcgacac cgactacaac 240
ggcaagttca agggcagagt gaccatcacc gccgacaagt ccacctccac cgcctacatg 300
gaactgtcct ccctgcggag cgaggacacc gccgtgtact actgcgcccg gaacgtgttc 360
gacggctact ggctggtgta ttggggccag ggcaccctcg tgaccgtgtc ctctgcttct 420
accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 480
gccgctctgg gctgcctcgt gaaggactac ttccccgagc ctgtgaccgt gtcctggaac 540
tctggcgctc tgacatccgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600
tactccctgt cctccgtcgt gaccgtgcct tccagctctc tgggcaccca gacctacatc 660
tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 720
tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780
gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840
acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900
gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 960
taccgggtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 1020
aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080
aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140
aagaaccagg tgtccctgtc ctgtgctgtg aaaggcttct acccctccga cattgccgtg 1200
gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260
tccgacggct cattcttcct ggtgagcaag ctgacagtgg acaagtcccg gtggcagcag 1320
ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380
tccctgtccc tgagccccgg caaatga 1407
<210> 37
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Anti-EGFR-Fc1 重链DNA序列
<400> 37
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctcag 60
gtccagctcc aggaaagcgg ccccggcctc gtcaaaccct ccgagacact ctccctcaca 120
tgcacagtct ccggcttctc cctcagcaac tacgacgtcc actgggtcag acaggccccc 180
ggcaaaggac tggaatggct cggcgtcatc tggtccggcg gaaacaccga ctacaacacc 240
ccattcacct ccaggctcac catctccgtg gacacctcca agaaccagtt ctccctcaaa 300
ctgagctccg tgaccgccgc cgacaccgct gtctattatt gcgccagagc cctcgactac 360
tacgactacg aattcgccta ctggggccag ggcaccctgg tgaccgtgtc atctgcttct 420
accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 480
gccgctctgg gctgcctcgt gaaggactac ttccccgagc ctgtgaccgt gtcctggaac 540
tctggcgctc tgacatccgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600
tactccctgt cctccgtcgt gaccgtgcct tccagctctc tgggcaccca gacctacatc 660
tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 720
tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780
gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840
acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900
gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 960
taccgggtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 1020
aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080
aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140
aagaaccagg tgtccctgag ctgtgcagtg aaaggcttct acccctccga cattgccgtg 1200
gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260
tccgacggct cattcttcct ggtgagcaag ctgacagtgg acaagtcccg gtggcagcag 1320
ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380
tccctgtccc tgagccccgg caaatga 1407
<210> 38
<211> 1410
<212> DNA
<213> Artificial Sequence
<220>
<223> Anti-Her2(T)-Fc1 重链DNA序列
<400> 38
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgag 60
gtgcagttgg tggagagcgg gggggggctg gtgcagcctg gaggaagttt gaggttgagc 120
tgtgccgcaa gcgggttcaa cattaaggac acatacattc actgggtgag gcaggcaccc 180
ggaaagggac tggagtgggt ggctaggatc taccccacca acggctacac aaggtacgcc 240
gacagtgtga agggccggtt caccatttcc gccgacacct ccaagaacac cgcctacctg 300
cagatgaaca gcctgagggc cgaggacacc gccgtctact actgctccag gtggggagga 360
gacggattct atgctatgga ctactgggga cagggcaccc tggtgaccgt gtcatctgct 420
tctaccaagg gcccctccgt gtttcctctg gccccttcca gcaagtccac ctctggcgga 480
acagccgctc tgggctgcct cgtgaaggac tacttccccg agcctgtgac cgtgtcctgg 540
aactctggcg ctctgacatc cggcgtgcac accttccctg ctgtgctgca gtctagcggc 600
ctgtactccc tgtcctccgt cgtgaccgtg ccttccagct ctctgggcac ccagacctac 660
atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagaaggt ggaacccaag 720
tcctgcgaca agacccacac ctgtccccct tgtcctgccc ctgaactgct gggcggacct 780
tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 840
gtgacctgcg tggtggtgga tgtgtcccac gaggaccctg aagtgaagtt caattggtac 900
gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc 960
acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag 1020
tacaagtgca aggtgtccaa caaggccctg cctgccccca tcgaaaagac catctccaag 1080
gccaagggcc agccccggga accccaggtg tacacactgc cccctagcag ggacgagctg 1140
accaagaacc aggtgtccct gagctgtgca gtgaaaggct tctacccctc cgacattgcc 1200
gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 1260
gactccgacg gctcattctt cctggtgagc aagctgacag tggacaagtc ccggtggcag 1320
cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1380
aagtccctgt ccctgagccc cggcaaatga 1410
<210> 39
<211> 705
<212> DNA
<213> Artificial Sequence
<220>
<223> Anti-Her2(T)-Fc1 轻链DNA序列
<400> 39
atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60
gacattcaga tgacccagag cccctcctcc ctctccgcct ccgtgggaga cagagttacc 120
atcacctgca gggcctccca ggacgtgaac accgccgtgg cctggtacca gcagaaaccc 180
ggcaaagccc ccaaactgct catctactcc gcctcatttc tgtacagcgg cgtgccctcc 240
cgcttctccg gttccagatc cggcaccgac ttcaccctga ctatctcctc cctccagccc 300
gaagacttcg ccacctacta ctgccagcag cactacacca ccccccccac cttcggccag 360
ggcacaaagg tcgaaatcaa gagaaccgtg gccgctccct ccgtgttcat cttcccacct 420
tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 540
gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 600
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 660
ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga 705
<210> 40
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Anti-Her2(P)-Fc1重链DNA序列
<400> 40
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgag 60
gtgcagttgg tggagagcgg gggggggctg gtgcagcctg gaggaagttt gaggttgagc 120
tgtgccgcaa gcgggttcac atttacagac tacacaatgg actgggtgag gcaggcaccc 180
ggaaagggac tggagtgggt ggctgatgtg aatcccaata gcggagggag catttacaac 240
cagagattca aggggcggtt caccttgtcc gtggacagga gcaagaacac actgtacctg 300
cagatgaaca gcctgagggc cgaggatacc gccgtctact attgcgccag gaacctcgga 360
ccctccttct attttgacta ctggggccag ggaaccctgg tgaccgtgtc atctgcttct 420
accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 480
gccgctctgg gctgcctcgt gaaggactac ttccccgagc ctgtgaccgt gtcctggaac 540
tctggcgctc tgacatccgg cgtgcacacc ttccctgctg tgctgcagtc tagcggcctg 600
tactccctgt cctccgtcgt gaccgtgcct tccagctctc tgggcaccca gacctacatc 660
tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 720
tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 780
gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840
acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 900
gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 960
taccgggtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 1020
aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1080
aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc 1140
aagaaccagg tgtccctgag ctgtgcagtg aaaggcttct acccctccga cattgccgtg 1200
gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1260
tccgacggct cattcttcct ggtgagcaag ctgacagtgg acaagtcccg gtggcagcag 1320
ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380
tccctgtccc tgagccccgg caaatga 1407
<210> 41
<211> 705
<212> DNA
<213> Artificial Sequence
<220>
<223> Anti-Her2(P)-Fc1 轻链DNA序列
<400> 41
atgtctgtgc ctacccaggt gctgggactg ctgctgctgt ggctgacaga cgcccgctgt 60
gacattcaga tgacccagag cccctcctcc ctctccgcct ccgtgggaga cagagttacc 120
atcacctgca aagccagcca ggacgtgagc atcggcgtgg cctggtacca gcagaaaccc 180
ggcaaagccc ccaaactgct catttactcc gcctcatacc gttacaccgg cgttccctcc 240
cgcttcagcg gatccggctc cggaaccgac ttcaccctga ctatctcctc cctccagccc 300
gaagacttcg ccacctacta ctgccagcag tactacattt acccctacac cttcggccag 360
ggcaccaagg tggaaatcaa gagaaccgtg gccgctccct ccgtgttcat cttcccacct 420
tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 480
ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 540
gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 600
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 660
ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga 705
<210> 42
<211> 1404
<212> DNA
<213> Artificial Sequence
<220>
<223> Anti-PD-L1(Ate)-Fc1 重链DNA序列
<400> 42
atggaatgga gctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcattctgaa 60
gtgcagctgg tggaaagcgg cggcggcctg gtgcagccgg gcggcagcct gcgcctgagc 120
tgcgcggcga gcggctttac ctttagcgat agctggattc attgggtgcg ccaggcgccg 180
ggcaaaggcc tggaatgggt ggcgtggatt agcccgtatg gcggcagcac ctattatgcg 240
gatagcgtga aaggccgctt taccattagc gcggatacca gcaaaaacac cgcgtatctg 300
cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt attgcgcgcg ccgccattgg 360
ccgggcggct ttgattactg gggccagggc accctggtga ccgtgtcatc tgcttctacc 420
aagggcccct ccgtgtttcc tctggcccct tccagcaagt ccacctctgg cggaacagcc 480
gctctgggct gcctcgtgaa ggactacttc cccgagcctg tgaccgtgtc ctggaactct 540
ggcgctctga catccggcgt gcacaccttc cctgctgtgc tgcagtctag cggcctgtac 600
tccctgtcct ccgtcgtgac cgtgccttcc agctctctgg gcacccagac ctacatctgc 660
aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggaacc caagtcctgc 720
gacaagaccc acacctgtcc cccttgtcct gcccctgaac tgctgggcgg accttccgtg 780
ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 840
tgcgtggtgg tggatgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 900
ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa ctccacctac 960
cgggtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag 1020
tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaaa agaccatctc caaggccaag 1080
ggccagcccc gggaacccca ggtgtacaca ctgcccccta gcagggacga gctgaccaag 1140
aaccaggtgt ccctgagctg tgcagtgaaa ggcttctacc cctccgacat tgccgtggaa 1200
tgggagtcca acggccagcc tgagaacaac tacaagacca ccccccctgt gctggactcc 1260
gacggctcat tcttcctggt gagcaagctg acagtggaca agtcccggtg gcagcagggc 1320
aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1380
ctgtccctga gccccggcaa atga 1404
<210> 43
<211> 1110
<212> DNA
<213> Artificial Sequence
<220>
<223> D1m-Fc2 DNA序列
<400> 43
atggagtgga gctgggtgtt cttgttcttc ttgtccgtga ccaccggggt gcacagcgag 60
gaggagttgc agatcatcca gcctgacaag agcgtgagcg tggccgccgg ggagagcgct 120
attctgcact gtaccatcac ctccctcttc cccgtgggcc ccattcagtg gttcagggga 180
gccgggcccg ccagagttct gatttacaac cagaggcagg gcccctttcc ccgggttacc 240
actgtctctg agaccaccaa gcgggagaac atggatttca gcatctccat cagcaacatt 300
actcccgccg acgccggcac ctactactgc atcaaattca gaaagggctc tcccgacacc 360
gaattcaaaa gcggcgccgg caccgaactg tccgtgcgag ctaagccctc cgagcccaaa 420
tcctcagaca agacccacac ctgtccccct tgtcctgccc ctgaactgct gggcggacct 480
tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 540
gtgacctgcg tggtggtgga tgtgtcccac gaggaccctg aagtgaagtt caattggtac 600
gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc 660
acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag 720
tacaagtgca aggtgtccaa caaggccctg cctgccccca tcgaaaagac catctccaag 780
gccaagggcc agccccggga accccaggtg tacacactgc cccctagcag ggacgagctg 840
accaagaacc aggtgtccct gtggtgtctc gtgaaaggct tctacccctc cgacattgcc 900
gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 960
gactccgacg gctcattctt cctgtacagc aagctgacag tggacaagtc ccggtggcag 1020
cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1080
aagtccctgt ccctgagccc cggcaaatga 1110
<210> 44
<211> 462
<212> PRT
<213> Artificial Sequence
<220>
<223> D1m-D2-Fc2 氨基酸序列
<400> 44
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val
20 25 30
Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser
35 40 45
Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala
50 55 60
Arg Val Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr
65 70 75 80
Thr Val Ser Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser
85 90 95
Ile Ser Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys
100 105 110
Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
115 120 125
Glu Leu Ser Val Arg Ala Lys Pro Ser Glu Pro Lys Ser Ser Gly Pro
130 135 140
Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser
145 150 155 160
His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly
165 170 175
Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser
180 185 190
Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu
195 200 205
Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln
210 215 220
Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Asp Lys Thr His Thr
225 230 235 240
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
245 250 255
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
260 265 270
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
275 280 285
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
290 295 300
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
305 310 315 320
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
325 330 335
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
340 345 350
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
355 360 365
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
370 375 380
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
385 390 395 400
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
405 410 415
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
420 425 430
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
435 440 445
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460