CN114195901A - 双特异性重组蛋白及其用途 - Google Patents

双特异性重组蛋白及其用途 Download PDF

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CN114195901A
CN114195901A CN202111094081.2A CN202111094081A CN114195901A CN 114195901 A CN114195901 A CN 114195901A CN 202111094081 A CN202111094081 A CN 202111094081A CN 114195901 A CN114195901 A CN 114195901A
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宋利平
王素琴
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Shanghai Linxizhi Enterprise Management Co ltd
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Shanghai Linxizhi Enterprise Management Co ltd
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Abstract

本发明公开了一种双特异性重组蛋白，包括第一功能结合片段、第二功能结合片段和Fc区；该双特异性重组蛋白靶向目的抗原的第一功能结合片段包含抗原结合片段，其中抗原结合片段中CL结构域的C末端或CH1结构域的C末端与具有免疫调节和/或代谢调节和/或内分泌调节功能的第二功能结合片段直接连接或通过接头序列连接。此外，还公开了该双特异性重组蛋白在制备治疗肿瘤、自身免疫性疾病、感染疾病、败血症、移植物抗宿主病、代谢紊乱、内分泌紊乱药物中的应用。本发明可显著提高双特异性重组蛋白第二功能结合片段的目标靶向性，定向调节作用，还能显著降低制备过程中生成的含有第二功能结合片段的非目标蛋白靶向非目标脏器、组织、细胞而引起的毒副作用。

Description

双特异性重组蛋白及其用途
技术领域
本发明属于生物医药领域，具体涉及一种双特异性重组蛋白及其用途。
背景技术
免疫是生命体的一种生理功能，依靠这种功能识别“自己”和“非己”成分，从而破坏和排斥进入人体的抗原物质(如病菌等)，或生命体自身所产生的损伤细胞和肿瘤细胞等，抵御肿瘤的发生发展，以维持生命体的健康。然而，异变细胞在某些情况下能通过多种机制逃避机体的免疫监视，在体内迅速增殖，形成肿瘤。人体也需要通过免疫抑制调节，避免免疫功能异常或持续活化，通过免疫炎症、细胞因子风暴、异体移植免疫排异等导致脏器损伤，比如自身免疫性疾病、败血病、移植物抗宿主病(GVHD)等。
肿瘤免疫治疗是通过重新启动并维持肿瘤-免疫循环，恢复机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种治疗方法。阻断这些免疫逃避机制，或是通过调节控制免疫调节和免疫激活机制来“促进”免疫激活是目前较为常用的肿瘤免疫治疗手段，例如抗PD-1抗体(如nivolumab、pembrolizumab)、抗PD-L1抗体(如durvalumab、atezolizumab)、抗CD47抗体/融合蛋白(如magrolimab、TTI-621)。
自身免疫性疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病，包括器官特异性自身免疫病和系统性自身免疫病，其中器官特异性自身免疫病主要有主要有慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等，系统性自身免疫病常见有系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、天疱疮、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎等。
败血症是指各种致病菌侵入血液循环，并在血中生长繁殖，产生毒素而发生的急性全身性感染，临床表现一般为急性起病、寒战、高热、呼吸急促、心动过速，以及皮疹、关节肿痛、肝脾肿大和精神、神志改变等，严重者可出现急性器官功能障碍，进一步加重后可发展为感染性休克、弥散性血管内凝血(DIC)和多器官功能衰竭。败血症是重症监护病房中最常见的致死原因之一。一方面，致病菌或条件致病菌在血中快速繁殖，免疫系统不能及时清除而发生急性全身性感染。另一方面，这些病菌分泌的毒素如脂多糖(LPS)可激活巨噬细胞，引起TNF、IL-1β、HMGB1等炎症因子的释放，从而产生系统性炎症反应，进一步加重败血症的病情，带来生命危险。
移植物抗宿主病(GVHD)是由于移植后异体供者移植物中的T淋巴细胞，经受者发动的一系列“细胞因子风暴”刺激，大大增强了其对受者抗原的免疫反应，以受者靶细胞为目标发动细胞毒攻击，急性移植物抗宿主病的发生率为30％～45％。
大量研究显示，免疫检查点或其配体、细胞因子等对疾病预防和治疗有非常重要的作用。
免疫检查点(checkpoint)指在免疫细胞上表达、能调节免疫激活程度的一系列分子，免疫检查点与其配体对调节自身免疫作用起着重要作用。免疫检查点分子的表达和功能异常是很多疾病发生的重要原因之一，例如抑制性免疫检查点分子过度表达或功能过强，免疫功能受到抑制，机体的免疫力就低下，人就容易受累于感染、肿瘤等疾病；反之，抑制性免疫检查点分子的免疫抑制功能如果太弱，机体的免疫功能则会异常活跃。肿瘤细胞也会表达一些物质，来激活抑制性免疫检查点，后者一旦被激活，就如同踩下“刹车”，使抗原不能被提呈至T细胞，阻断了肿瘤免疫环中的提呈抗原过程，从而抑制T细胞的免疫功能，使得肿瘤细胞逃脱监视、存活下来。常见的免疫检查点及其配体例如PD-1/PD-L1、LAG-3/MHCII、CTLA-4/B7-1或B7-2、TIM-3/Galectin-9、SIRPα/CD47、TIGIT/Nectin2或Nectin3、BTLA/HVEM等。
PD-1(程序性死亡受体1)，是一种重要的免疫抑制分子，为免疫球蛋白超家族，PD-1和PD-L1结合启动T细胞的程序性死亡，使肿瘤细胞获得免疫逃逸。因此，大量临床研究结果证明阻断PD-1与PD-L1相互作用产生的免疫调节作用对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义。
LAG-3(Lymphocyte-activation gene 3)属于免疫球蛋白超家族，由胞外区、跨膜区和胞质区3个部分组成。抑制LAG-3能够让T细胞重新获得细胞毒性，增强对肿瘤的杀伤效果，同时抑制LAG-3还能够降低调节T细胞抑制免疫反应的功能。
CTLA-4(细胞毒T淋巴细胞相关抗原4，cytotoxic T lymphocyte-associatedantigen-4,)又名CD152，是一种白细胞分化抗原，是T细胞上的一种跨膜受体，与CD28共同享有B7分子配体，CTLA-4以比CD28更大的亲和力和亲合力结合CD80和CD86(也称为B7-1和B7-2)，向T细胞传递抑制信号，参与免疫反应的负调节。
TIM-3又称HAVCR2，属于TIM基因家族。TIM-3作为一种负调控的免疫检查点，存在于不同类型的免疫细胞中，包括T细胞、调节性T细胞(Tregs)、树突状细胞(DC)、B细胞、巨噬细胞、自然杀伤细胞(NK)和肥大细胞。TIM-3是一种Ⅰ型膜蛋白，由281个氨基酸组成。它由一个胞外区、一个单跨膜结构域和一个C-末端细胞质尾部组成。TIM-3与其配体Gal-9通过负性调节T细胞免疫来抑制肿瘤免疫。TIM-3IgV结构域与Gal-9的连接可终止Th1免疫应答。TIM-3可诱导免疫耐受，与哮喘、食物过敏及自身免疫性疾病如多发性硬化症和类风湿性关节炎等有关。TIM-3还能抑制T细胞的免疫应答，并与导致慢性病毒感染的免疫耗竭有关。
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。
SIRP-alpha(SIRPα)是SIRP家族中的一个典型的抑制性免疫受体，可与体内广泛存在的跨膜糖蛋白CD47相互作用，传递抑制性信号。而SIRPα融合蛋白则可以通过竞争阻断CD47与巨噬细胞表面的SIRP-alpha结合，解除肿瘤细胞上CD47对巨噬细胞的抑制作用，恢复巨噬细胞免疫功能，发挥抗肿瘤作用。
TIGIT(含Ig及ITIM结域的T细胞免疫受体，又称VSlG9、VSTM3或WUCAM)，是免疫球蛋白脊髓灰质炎病毒受体家族CD28家族样受体的一员，在T细胞和自然杀伤(NK)细胞亚群上中表达。一般情况下，其表达水平很低，但当这些细胞激活后，其蛋白水平会上调。比如在肿瘤微环境中，肿瘤浸润淋巴细胞中的TIGIT经常处于高表达水平，TIGIT可与免疫细胞、非免疫细以及肿瘤细胞中的受体CD155(poliovirus receptor，PVR)、CD112(PVRL2,nectin-2)、CD113(Nectin-3)结合，结合后导致T细胞失活，T细胞和自然杀伤细胞的细胞毒性受到抑制。
BTLA是表达于活化T、B淋巴细胞的一个重要的免疫检查点分子，与其他免疫检查点分子如PD-1和CTLA-4具有类似的结构(单一的免疫球蛋白可变区IgV细胞外结构域)和相近的细胞内的信号传导机制(胞质内两个ITIM结构域募集活化SHP-1和SHP-2磷酸酶抑制淋巴细胞功能)。TNF受体家族的HVEM(Herpesvirus entry mediator，疱疹病毒进入中介体)已被鉴定为BTLA的配体，广泛地表达于人体免疫细胞，如T细胞、B细胞、NK细胞、髓样细胞和树突细胞，以及多种肿瘤细胞(包括非小细胞肺癌、黑色素瘤、结肠直肠癌和淋巴瘤)。在正常生理情况下，BTLA与其配体HVEM结合后，可以抑制体内淋巴细胞的过度活化，防止免疫系统对自身的损伤。但在肺癌、黑色素瘤、结肠直肠癌和淋巴瘤等肿瘤细胞通过高表达HVEM，与肿瘤特异的杀伤性淋巴细胞表达的BTLA结合后，可抑制淋巴细胞的免疫功能。HVEM的肿瘤高表达与不良预后相关联。
细胞因子(cytokine，CK)是由多种组织细胞(主要为免疫细胞)所合成和分泌的小分子多肽或糖蛋白。细胞因子能介导细胞间的相互作用，具有多种生物学功能，如调节细胞生长、分化成熟、功能维持、调节免疫应答、参与炎症反应、创伤愈合和肿瘤消长等。依据细胞因子的结构与功能，可以将细胞因子分为白介素超家族、干扰素家族、肿瘤坏死因子超家族、TGF-β超家族、趋化因子家族、集落刺激因子家族、生长因子家族等。
白介素，是由多种细胞产生并作用于多种细胞的一类细胞因子。由于最初是由白细胞产生又在白细胞间发挥作用，所以由此得名，现仍一直沿用。指一类分子结构和生物学功能已基本明确，具有重要调节作用而统一命名的细胞因子，它和血细胞生长因子同属细胞因子。两者相互协调，相互作用，共同完成造血和免疫调节功能。白细胞介素在传递信息，激活与调节免疫细胞，介导T、B细胞活化、增殖与分化及在炎症反应中起重要作用。
IL-2，是由多细胞来源(主要由活化T细胞产生)，又具有多向性作用的细胞因子，促进淋巴细胞生长、增殖、分化，对机体的免疫应答和抗病毒感染等有重要作用。IL-2能活化T细胞，促进细胞因子产生；刺激NK细胞增殖，增强NK杀伤活性及产生细胞因子，诱导LAK细胞产生；促进B细胞增殖和分泌抗体；激活巨噬细胞。
IL-10，也称为细胞因子合成抑制因子(CSIF)，具有双向免疫调节作用，IL-10可以通过抗原提呈细胞(APC)发挥免疫抑制，通过T细胞发挥负调节作用，在肿瘤环境中对免疫应答具有负向调节作用；另外，IL-10对T、B淋巴细胞具有刺激作用，并且在肿瘤环境中IL-10也能发挥刺激作用；IL-10的双向调节作用自从被发现起就一直被关注，它不仅影响免疫系统，而且可以通过调节生长因子、细胞因子影响许多病理生理过程，包括血管生成、肿瘤形成和感染，还能通过诱导调节性T细胞在外周耐受中建立作用；对克罗恩病、类风湿性关节炎银屑病、HCV感染、HIV感染等，IL-10都发挥着重要作用。天然状态下，IL-10通过形成同源二聚体与其受体结合形成复合物后激活下游信号通路，并发挥其生理学功效。2000年，Josephson等研究设计了IL-10突变体，其可在单体情况下结合IL-10受体并激活下游信号通路(Josephson,K.Design and analysis of an engineered human interleukin-10monomer.[J].Journal of Biological Chemistry,2000,275(18):13552-7.)。
干扰素，是一组具有多种功能的活性蛋白质，主要由单核细胞和淋巴细胞产生的细胞因子，分为三类，I型，II型，III型。I型干扰素以IFN-α与IFN-β为主由先天性免疫细胞分泌，II型干扰素即IFN-γ，主要由活化后的T细胞分泌产生，III型干扰素为几种IFN-λ。干扰素具有广谱的抗病毒、影响细胞生长，以及分化、调节免疫功能等多种生物活性，具体功能如下：
(1)抗病毒作用：I型干扰素是免疫系统中的主要抗病毒防御与调节因子。机体在早期的病毒感染期间，I型干扰素即可控制病毒的生长和增殖。它一方面直接激活免疫细胞，另一方面可间接抑制病毒的复制过程。
(2)抗菌作用：干扰素能通过下调转铁蛋白受体减少细菌供铁量或通过诱导产生内源性NO直接抑制细胞内细菌，还能增加单核巨噬细胞的吞噬小体——溶酶体溶解细菌作用，通过以上途径共同达到消灭细菌的作用。
(3)抗寄生虫作用：干扰素可激活巨噬细胞
Figure BDA0003268638110000051
活化的
Figure BDA0003268638110000052
可表达高水平的诱导型-氧化氮合酶(iNOS)催化L-精氨酸产生NO，NO对接种病原体有抑制和杀伤作用。并据报道，IFN-γ能激活
Figure BDA0003268638110000053
产生NO，同时促进NO合成作用受剂量依赖性，剂量越高作用越明显。Daubener等(2001)发现用IFN-γ刺激人脑微血管内皮细胞(HBMEC)能诱导其抗弓形虫病。IFN-γ刺激后的HBMEC能抑制弓形虫生长，提高TNF-α的出现，这与IDO的活性有关。另外，在HBMEC的培养中加入过量的色氨酸能完全抑制IFN-γ-TNF-α介导的抗弓形虫病，表明IDO能介导其保护性，并且据报道，IFN-γ依赖IDO的表达而起作用。
(4)参与免疫调节：参与免疫调节的主要为IFN-γ，又称为免疫调节作用干扰素。免疫调节干扰素可对IgG的Fc受体表达，从而有利于巨噬细胞对抗原的吞噬，K、NK细胞对靶细胞的杀伤以及T、B淋巴细胞的激活，增强机体免疫应答能力。IFN-γ可使巨噬细胞表面MHCⅡ类分子的表达增加，增强其抗原递呈能力。此外还可以通过增强巨噬细胞表面表达Fc受体，促进巨噬细胞吞噬免疫复合物、抗体包被的病原体和肿瘤细胞。同时还可以刺激中性粒细胞，增强其吞噬能力，活化NK细胞，增强其细胞毒作用等作用来参与免疫调节。
(5)抗肿瘤作用：IFN-α、IFN-β具有广泛的抗肿瘤作用，IFN-γ对机体免疫反应有多方面的调节作用，能激活效应细胞，提高自然杀伤细胞、巨噬细胞和肿瘤浸润淋巴细胞的活性，促进单核细胞循环，增强免疫细胞表面抗原和抗体的表达，刺激IL-2、肿瘤坏死因子、IFN-α等细胞因子的产生，抑制肿瘤细胞分裂，诱导基因全成抗病毒蛋白等。
肿瘤坏死因子超家族，肿瘤坏死因子(TNF)是在能够杀死小鼠中的癌细胞的血清中发现的细胞因子。成员主要包括TNF-α、TNF-β、淋巴毒素-β、CD40L、FasL、CD30L、4-1BBL、CD27L和OX40L，TNF相关凋亡诱导配体(TRAIL)，LIGHT受体激活剂(RANKL)，TNF相关的凋亡诱导因子(TWEAK)，增殖诱导配体(APRIL)，B细胞激活因子(BAFF)，血管内皮细胞生长抑制剂(VEGI)，胞质素A(EDA-A1，EDA-A2)和糖皮质激素诱导的肿瘤坏死因子受体相关配体(GITRL)。肿瘤坏死因子TNF超家族成员与数十个受体特异性识别，构成配体-受体作用系统。TNF受体(TNFR)主要是跨膜蛋白，参与一些生理过程，如宿主防御、炎症、细胞凋亡、自身免疫、以及免疫、外胚层和神经系统的发育和器官发生等。TNF引起肿瘤细胞坏死(细胞肿胀、细胞器破坏、细胞裂解)和细胞凋亡(细胞收缩、凝聚体形成、DNA断裂)，在各种免疫和炎症过程中起关键作用。
集落刺激因子(CSF)，可刺激骨髓未成熟细胞分化成熟并在体外可刺激集落形成的细胞因子。根据细胞因子刺激不同造血细胞系或不同分化阶段的细胞在半固体培养基中形成不同细胞集落，分别命名为粒细胞CSF(G-CSF)、巨噬细胞CSF(M-CSF)、粒细胞和巨噬细胞CSF(GM-CSF)、多重集落刺激因子(multi-CSF，又称IL-3)、干细胞因子(SCF)、红细胞生成素(EPO)。它们对不同发育阶段的造血干细胞起促增殖、分化的作用，是血细胞发生必不可少的刺激因子。广义上，凡是刺激造血的细胞因子都可统称为CSF，例如可刺激胚胎干细胞的白血病抑制因子(leukemia inhibitory factor,LIF)以及刺激血小板的血小板生成素(thrombopoietin)等均有集落刺激活性。此外，CSF也作用于多种成熟的细胞，促进其功能具有多相性的作用。
趋化因子，是一类由细胞分泌的小细胞因子或信号蛋白。由于它们具有诱导附近反应细胞定向趋化的能力，因而命名为趋化因子。趋化因子蛋白的共同结构特征包括，分子量小(约8-10kDa)，有四个位置保守的半胱氨酸残基以保证其三级结构。趋化因子被分为四个主要亚家族：CXC、CC、CX3C和XC。所有这些蛋白都通过与G蛋白连接的跨膜受体(称为趋化因子受体)相互作用来发挥其生物学效应这些蛋白质结合到趋化因子受体而起作用，趋化因子受体是G蛋白偶连的跨膜受体，选择性地表达在靶细胞表面。
趋化因子的主要功能是在炎症和体内平衡过程中管理白细胞向各自位置的迁移(归巢)。基础归巢作用：在胸腺和淋巴组织中产生的基础的稳态趋化因子。趋化因子CCL19和CCL21(在淋巴结和淋巴管内皮细胞中表达)及其受体CCR7(在细胞中注定要归巢到这些器官的细胞中表达)是它们在归巢中的稳态功能的最好例证。利用这些配体可使抗原呈递细胞(APC)在适应性免疫应答过程中转移至淋巴结。其他的稳态趋化因子受体包括：CCR9、CCR10和CXCR5，它们作为细胞地址的一部分对于组织特异性的白细胞归巢非常重要。CCR9支持白细胞向肠内迁移，CCR10支持皮肤迁移，CXCR5支持b细胞向淋巴结滤泡迁移。骨髓中生成的CXCL12(SDF-1)促进了骨髓微环境中B祖细胞的增殖。炎症归巢作用：炎症趋化因子在感染或损伤过程中产生高浓度，并决定炎症性白细胞向受损区域的迁移。典型的炎性趋化因子包括：CCL2、CCL3和CCL5、CXCL1、CXCL2和CXCL8。
一部分趋化因子被认为是促炎性细胞因子，可以在免疫应答过程中诱导免疫系统的细胞进入感染部位。而有些趋化因子被认为维持机体自我调节，在正常的组织维持或发育过程中控制细胞的迁徙。
趋化因子根据其趋化作用的细胞类型不同，可以分为如下几类：
单核/巨噬细胞趋化因子:吸引单核/巨噬细胞到炎症部位的关键趋化因子包括:CCL2、CCL3、CCL5、CCL7、CCL8、CCL13、CCL17和CCL22。
T淋巴细胞趋化因子：参与T淋巴细胞募集到炎症部位的四个关键趋化因子是:CCL2、CCL1、CCL22和CCL17。此外，T细胞激活后诱导CXCR3表达，活化的T细胞被炎症部位吸引，在炎症部位分泌IFN-γ诱导的趋化因子CXCL9、CXCL10和CXCL11。
肥大细胞趋化因子：表面表达多种趋化因子受体：CCR1、CCR2、CCR3、CCR4、CCR5、CXCR2、CXCR4。这些受体CCL2和CCL5的配体在肺肥大细胞募集和活化中起重要作用。也有证据表明CXCL8可能抑制肥大细胞。
嗜酸性粒细胞趋化因子：嗜酸性粒细胞向各种组织的迁移涉及CC家族的几种趋化因子：CCL11、CCL24、CCL26、CCL5、CCL7、CCL13和CCL3。趋化因子CCL11(eotaxin)和CCL5(rantes)通过嗜酸性粒细胞表面的一个特定受体CCR3发挥作用，而嗜酸性粒细胞在最初募集到病变中发挥重要作用。
中性粒细胞趋化因子：主要由CXC趋化因子调节。例如，CXCL8(IL-8)是中性粒细胞的趋化剂，并激活其代谢和脱颗粒。
TGF-β超家族，是一组调节细胞生长和分化的细胞因子。这一家族除TGF-β外，还有活化素(activins)、抑制素(inhibins)、缪勒氏管抑制质(Mullerian inhibitorsubstance,MIS)和骨形成蛋白(bone morpho-genetic proteins,BMPs)。TGF-β在炎症、组织修复和胚胎发育等方面发挥作用，对细胞的生长、分化和免疫功能都有重要的调节作用，具体功能如下：
(1)抑制免疫活性细胞的增殖：①抑制IL-3、GM-CSF、M-CSF所诱导小鼠造血前体细胞和LTBMC的集落形成，并降低巨核细胞对IL-3T和CSF的反应性。②抑制ConA诱导或ConA与IL-2、IL-6联合诱导的胸腺细胞增殖。③抑制丝裂原、同种异体抗原刺激的T细胞增殖或IL-2依赖的T细胞生长。④抑制SAC刺激后IL-2依赖的B细胞增殖。
(2)对细胞表型的调节：①抑制IL-2诱导的T细胞IL-2R、TfR和TLiSA1活化抗原的表达，对CD3表达未见有影响。②抑制IFN-γ诱导黑素瘤细胞MHCⅡ类抗原表达。
(3)抑制淋巴细胞的分化：①抑制IL-2和BCDF依赖的B细胞分泌IgM，促进B细胞分泌Ig类型转换为IgA和IgE。②抑制混合淋巴细胞培养(MLC)中CTL、NK和LAK功能，这种抑制作用可被TNF-α(小鼠MIC)或IL-2(人MLC)所逆转。③抑制PBMC中NK活性。④抑制ConA和IL-2、IL-6协同诱导小鼠胸腺MHC非限制杀伤性细胞的活性。
(4)抑制细胞因子产生：如抑制PBMC中IFN-γ和TNF-α的产生。
(5)其它调节作用：①促进成纤维细胞、成骨细胞和雪旺氏细胞的生长。TGF-β1、TGF-β2促进人成纤维细胞IL-6的产生，其机理可能是通过对IL-6基因转录的调节。②抑制上皮细胞、破骨细胞、内皮细胞生长和脂肪、心肌、骨骼肌的形成。TGF-β可拮抗EGF的某些生物学功能。③促进细胞外基质(ECM)如胶原蛋白、纤粘连蛋白的表达和抑制ECM的降解，对细胞的形态发生、增殖和分化过程起着重要作用，有利于胚胎发育和细胞修复。动物体内实验表明，局部注射TGF-β可以促进伤口愈合和典型肉芽组织形成。④单核细胞和成纤维细胞的趋化剂，但不引起胶颗粒和氧化物的产生。⑤抑制淋巴细胞与内皮细胞的黏附。⑥促进嗜碱性粒细胞释放组织胺。
(6)TGF-β1与原癌基因表达：TGF-β1能诱导c-sis的表达，但抑制c-myc的表达，这种诱导或抑制作用与作用细胞种类及TGF-β的不同功能有关。如TGF-β诱导成纤维细胞中c-sis基因表达，与促进其在软琼脂中生长有关；而对上皮角朊细胞生长的抑制则与抑制c-myc基因表达有关。TGF-β1、TGF-β2和TGF-β3在大多数生物学作用方面非常相似，但在有些作用方面可有很大差异，如TGF-β2对血管内皮细胞和造血祖细胞的生长抑制作用仅为TGF-β1和TGF-β3的1％。
TGF-β在治疗伤口愈合，促进软骨和骨修复以及通过免疫抑制治疗自身免疫性疾病和移植排斥等方面有潜在的应用前景。
生长因子，一类由多种细胞分泌，作用于特定的靶细胞，调节细胞分裂、基质合成与组织分化的细胞因子。生长因子有多种，如血小板类生长因子(血小板来源生长因子，PDGF；骨肉瘤来源生长因子ODGF)、表皮生长因子(EGF)、成纤维细胞生长因子(αFGF、βFGF)、类胰岛素生长因子(IGF-Ⅰ、IGF-Ⅱ)、神经生长因子(NGF)等。
然而，免疫调节往往是系统性的，免疫激活常常伴随免疫系统过度活化，导致如下免疫相关毒性反应：
皮肤：表现为皮疹/斑丘疹、瘙痒、大疱性皮炎综合征等；
内分泌：表现为甲状腺功能减退、甲状腺功能亢进、原发性肾上腺功能减退、高血糖等；
肝脏：主要表现为转氨酶升高；
胃肠道：主要表现为腹泻/结肠炎；
肺脏：免疫相关性肺炎；
还有其他相对少见的不良反应，包括神经毒性、血液毒性、肾脏毒性、心脏毒性、眼毒性等。
靶向免疫检查点或其配体、细胞因子或其受体的治疗已展示出对肿瘤、自身免疫性疾病、败血症、移植物抗宿主病(GVHD)等疾病的治疗效果，但药物本身带来的潜在的相关毒性反应限制了药物剂量，降低了药效。此外，免疫抑制又常常伴随免疫系统过度低下，导致易发细菌、病毒、真菌等感染，导致炎症反复发作，诱发肿瘤发生。
因此，单一的靶点治疗，如各种细胞因子、单抗、配体/受体Fc融合蛋白等，往往不足以达到最佳的安全窗与治疗效果，两个或多个靶点协同作用的治疗概念应运而生。在这种情况下，靶向两个或多个靶点的双特异性抗体/融合蛋白(双抗分子)或多特异性抗体/融合蛋白(多抗分子)可能提供单抗难以实现的新的治疗应用。其中就包括分别靶向目标细胞表面靶抗原(如CD19、HER2等)和靶向免疫调节靶点(如CD3、PD-1、CD47等)或携带免疫调节剂(如IL-2、4-1BB、SIRPα、CD80等)的双/多特异性抗体/融合蛋白的设计思路和临床应用(如Blinatumomab，博纳吐单抗)。
常见的对称结构双抗分子/双特异性融合蛋白，虽然具有一定的靶向聚集效应，使药物分布在靶器官得到一定程度的累积，且药学开发工艺、特别是纯化工艺相对可控，但其结构仍然存在一定的限制性，特别是靶器官与非靶器官的药物浓度差异并不十分显著，且如果采用皮下或静脉等非靶器官给药，血液中药物浓度依然是导致免疫毒性、制约其安全剂量的主要问题。因此靶向靶抗原的抗原结合片段/融合蛋白与靶向免疫检查点/细胞因子受体的抗原结合片段/融合蛋白的亲和力的组合需要严格把控，以免在双特异性分子其靶向靶抗原部分未达到靶细胞最佳浓度富集效果时，靶向免疫调节的部分已引发免疫相关毒副作用。
除了对称性结构，也有双特异性抗体/融合蛋白通过不对称结构形式，依托左右臂的亲和力差异，可进一步提高药物在靶向抗原阳性的细胞表面富集，从而进一步降低靶向免疫调节部分引发的免疫相关毒副作用。然而，不对称结构除了存在药学工艺难度提高、收率降低、生产成本增加等不利因素外，免疫调节臂单体或聚体杂质则成为引发免疫相关不良事件的重要风险来源。以TTI-621(Trillium Therapeutics Inc.开发的SIRPα-Fc融合蛋白)为例，根据TTI-621已公开的临床研究结果，在极低的剂量(0.2mg/kg)下即引起20％以上患者三级及以上血小板降低的严重副作用(因TTI-621结合血小板上高表达的CD47导致血小板被免疫细胞清除)。而如CN108864290A中说明书附图3所示的双抗结构，制备过程中其成品将潜在存在右臂单体/二聚体结构(即TTI-621类似物)的杂质，按5mg/kg-20mg/kg剂量的常规肿瘤靶向治疗类抗体药物的用药剂量，在极低的杂质浓度(1-4％)即可达到TTI-621严重毒副作用的剂量浓度，因此该结构对成品的杂质含量控制极其严苛，生产成本将严重上升。
此外，部分免疫调节的融合蛋白如干扰素，因其产品特性，冻融稳定性一般较差，如重组人白蛋白干扰素-α2b融合蛋白(rHSA-IFN-α2b)反复冻融后聚合体逐渐增加，冻融4次后聚合体含量达到17.91％，重组人白蛋白干扰素-α2b融合蛋白不宜冻融(夏毅等，重组人白蛋白干扰素α-2b融合蛋白的稳定性研究，生物学杂志，2008，25(006):38-40)，以至于对生产、运输及使用的限制较多。
因此，亟待发明一种可以同时靶向目的抗原并发挥免疫调节作用的双抗或双特异性结构，该结构不仅可以实现靶细胞高浓度富集并发挥免疫调节作用，更可在血液中即使达到较高的浓度，也不会在没有目的抗原存在的情况下，单纯依靠免疫调节端发挥出免疫调节功能，从而实现更精准目标脏器、组织、细胞的免疫调节，降低免疫相关毒副作用风险，同时提高产品的稳定性(例如冻融稳定性)。
发明内容
本发明要解决的技术问题之一是提供一种对含目的抗原的目标靶细胞有强靶向性，同时不结合或弱结合不含有目的抗原的非目标靶细胞的具有定向调节作用的双特异性重组蛋白，显著提高重组蛋白第二功能结合片段对目标靶细胞的靶向性，定向调节免疫作用，同时显著降低制备过程中生成的含有第二功能结合片段的潜在风险杂质靶向含有非目标靶细胞的脏器、组织等而引起的毒副作用。
本发明要解决的技术问题之二是提供该具有定向调节作用的双特异性重组蛋白在制药中的用途。
本发明要解决的技术问题三是提供一种可以提高产品稳定性(如冻融稳定性)的双特异性重组蛋白，解决部分免疫调节融合蛋白(如干扰素等)因其产品自身特性带来的产品不稳定性及因此对生产、运输、及使用的限制要求。
在一个方面，本发明提供一种双特异性重组蛋白，所述双特异性重组蛋白包括第一功能结合片段、第二功能结合片段和Fc区；所述双特异性重组蛋白靶向目的抗原的第一功能结合片段包含抗原结合片段，其中抗原结合片段中CL结构域的C末端或CH1结构域的C末端与具有免疫调节功能和/或代谢调节功能和/或内分泌调节功能的第二功能结合片段直接连接或通过接头序列连接。
优选地，所述抗原结合片段与所述第二功能结合片段的N端直接连接或通过接头序列连接；并且，所述第二功能结合片段的C端直接连接或通过接头序列连接Fc的N端。
在一个实施方案中，所述具有免疫调节功能的第二功能结合片段靶向免疫检查点、免疫检查点配体或细胞因子受体。可选地，所述具有免疫调节功能的第二功能结合片段靶向PD-1或其配体、CD47或其配体、CD24或其配体、干扰素受体(如I型或II型干扰素受体)、白介素受体。
在一个实施方案中，所述具有代谢调节功能的第二功能结合片段靶向代谢调节因子、代谢调节因子受体。可选地，所述具有代谢调节功能的第二功能结合片段靶向胰岛素受体、成纤维细胞生长因子受体。
在一个实施方案中，所述具有内分泌调节功能的第二功能结合片段靶向内分泌调节因子、内分泌调节因子受体。可选地，所述具有内分泌调节功能的第二功能结合片段靶向激素受体。
在一个实施方案中，所述抗原结合片段中可变区(V区)和恒定区(C区)直接连接或通过接头序列连接；或所述抗原结合片段与Fc区直接连接或通过接头序列连接；或同时使用上述两种方式连接。
在一个实施方案中，所述接头序列包含(GGGGS)n、(GGGS)n、(GGS)n、(G)n、(GS)n、(EAAAK)n、或(XP)n，n为自然数。优选地，所述n为0-5的自然数。
在一个实施方案中，接头序列为(GGGGS)n，n＝0、1、2、3、4、5。
在一个实施方案中，所述第二功能结合片段结合细胞因子受体或免疫检查点或免疫检查点配体。优选地，所述第二功能结合片段为细胞因子或免疫检查点配体或免疫检查点配体结合蛋白，或其功能片段或其突变体；可选为，人源SIRP家族胞外功能片段、人干扰素家族功能片段、肿瘤坏死因子超家族功能片段、TGF-β超家族功能片段、白介素类功能片段、趋化因子家族功能片段、集落刺激因子家族功能片段、生长因子功能片段或其突变体。
在一个实施方案中，所述第二功能结合片段为人源SIRPα胞外D1结构域或其突变体。优选地，该突变体为低亲和突变体，即该突变体与人CD47蛋白的结合亲和力不高于野生型蛋白与人CD47蛋白的结合亲和力。
在一个实施方案中，所述第二功能结合片段为人干扰素γ(IFN-γ)或人干扰素α(IFN-α)或人干扰素β(IFN-β)、其截短体或其突变体。
在一个实施方案中，所述第二功能结合片段为白介素、其截短体，或其突变体。
在一个实施方案中，所述白介素为免疫调节或趋化因子，选自以下任意一种：IL-1家族、IL-2家族、IL-3家族、IL-6家族、IL-8家族、IL-10家族、IL-12家族和IL-17家族。优选的，所述第二功能结合片段为人源IL-10单体突变体(氨基酸序列如SEQ ID NO:52所示)，人源IL-12A或其突变体(氨基酸序列如SEQ ID NO:51所示)、由人源IL-15(氨基酸序列如SEQID NO:53所示)和IL-15RαSUSHI(氨基酸序列如SEQ ID NO:54所示)直接连接或通过接头序列连接组成的融合蛋白或其突变体。
在一个实施方案中，所述第一功能结合片段靶向选自下列靶标中的任意一种或多种：5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGFR、EGFRvIII、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、Notch 1、PD-L1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TIGIT、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、BCMA、CD138、TROP2、Siglec15、CD155和AFP。
较佳地，当所述第二功能结合片段包含人源SIRPα胞外D1结构域或其突变体时，第一功能结合片段靶向肿瘤细胞或免疫细胞。
当所述第二功能结合片段为人干扰素α(IFN-α)或人干扰素β(IFN-β)或人干扰素γ(IFN-γ)、其截短体或其突变体时，第一功能结合片段靶向肿瘤细胞或免疫细胞。
当所述第二功能结合片段为白介素、其截短体或突变体时，所述第一功能结合片段靶向肿瘤细胞或免疫细胞。
在一个实施方案中，所述双特异性重组蛋白由A链和B链组成，所述A链与B链通过分子间作用力结合，或通过共价键例如链间二硫键结合，或通过盐键结合，或通过上述结合方式中的两种或三种的组合而结合。所述A链是由VH、CL/CH1、CH2、CH3结构域顺序组成的蛋白，或由VL、CL/CH1、CH2、CH3结构域顺序组成的蛋白，结构域之间可直接连接或通过接头序列(Linker)连接；所述B链是由VL、CL/CH1、第二功能结合片段、CH2、CH3结构域顺序组成的蛋白，或由VH、CL/CH1、第二功能结合片段、CH2、CH3结构域顺序组成的蛋白，结构域之间可直接连接或通过接头序列(Linker)连接；例如，当“A链”为VH-CH1-CH2-CH3时，则“B链”为VL-CL-第二功能结合片段-CH2-CH3；当“A链”为VL-CL-CH2-CH3时，则“B链”为VH-CH1-第二功能结合片段-CH2-CH3，当“A链”为VH-CL-CH2-CH3时，则“B链”为VL-CH1-第二功能结合片段-CH2-CH3；当“A链”为VL-CH1-CH2-CH3时，则“B链”为VH-CL-第二功能结合片段-CH2-CH3。上述CH2的N端还含有铰链区。
在一个实施方案中，所述双特异性重组蛋白的Fc区包含Fc区天然序列或Fc非天然序列；更优选地，所述Fc区为人Fc区；进一步更优选地，其中A链与B链的Fc区是通过knobs-into-holes结合的。
在一个实施方案中，所述所述Fc区为人IgG的Fc区；优选地，所述Fc区为人IgG1或IgG4的Fc区。
在一个实施方案中，所述第一功能结合片段的CL结构域的C末端或CH1结构域的C末端或第二功能结合片段的C末端与所述Fc区直接连接或通过接头序列连接。
在一个实施方案中，所述第一功能结合片段为抗原结合片段，任选地为人鼠嵌合抗原结合片段、人源抗原结合片段、全人源抗原结合片段；所述第一功能结构片段选自下列靶标抗体的抗原结合片段中的任意一种或多种：5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD71、CD74、CD79b、CD80、CD83、CD86、CD98、CD206、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CCR5、CXCR4、DLL-4、EGFR、EGFRvIII、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、Notch 1、PD-L1、PD-L2、PD-1、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、TIGIT、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、BCMA、CD138、TROP2、Siglec15、CD155和AFP；优选地所述第一功能结合片段为人源化或全人源抗体的抗原结合片段。
在一个实施方案中，所述第一功能结合片段靶向CD20、EGFR、EGFRvIII、PD-L1、PD-L2、HER2、HER3、CD138、CD44、CD24、EpCAM、CLDN18.2、CD38、BCMA、MUC1、或TROP2时，所述第二功能结合片段包含SIRPα胞外D1结构域或其突变体。优选地，所述第二功能结合片段如SEQID NO:50所示。
在一个实施方案中，所述第一功能结合片段靶向Siglec15、PD-L1、PD-L2、CD71、CD80、CD86、CD206、CCR5时，所述第二功能结合片段包含IFN-α或IFN-β或IFN-γ或IL-10单体突变体或IL-12A或IL-15与IL-15RαSUSHI形成的融合蛋白或上述细胞因子的截短体或保持细胞因子功能的突变体。优选地，该突变体对相应细胞因子受体的活性不高于野生型细胞因子与对应细胞因子受体的结合亲和力。更优选地，所述第二功能结合片段所包含的细胞因子如SEQ ID NO:51、或SEQ ID NO:52、或SEQ ID NO.53与SEQ ID NO:54形成的融合蛋白。
在一个实施方案中，所述第一功能结合片段为抗CD20抗体奥法木单抗(Ofatumumab)，或抗EGFR抗体帕尼单抗(Panitumumab)，或抗EpCAM抗体(Catumaxomab)，或抗CD24抗体SWA11的抗原结合片段，所述第二功能结合片段为人源SIRPα胞外D1结构域(如SEQ ID NO:50所示)。
在一个实施方案中，所述第一功能结合片段包含抗甲胎蛋白(AFP)抗体tacatuzumab的抗原结合片段，所述第二功能结合片段为人源IFN-α2b或人源IFNβ(SEQ IDNO:55)。
在一个实施例中，所述双特异性重组蛋白A链和B链是通过IgG Fc相结合的，优选地，通过knobs-into-holes结合的。例如，所述knobs-into-holes为由T366W突变形成突出的“knobs”型，和1个氨基酸突变(Y407V)形成凹陷的“holes”型或3个氨基酸突变(T366S、L368A和Y407V)形成凹陷的“holes”型。突变根据Kabat编号方案[Eu numbering scheme ofKabat et al.(1991)]，其从左至右分别显示为原氨基酸残基、突变位点和取代氨基酸残基，例如T366W中，T为原氨基酸残基、366为突变位点和W为取代T的氨基酸残基。
在一些实施例中，所述第一功能结合片段靶向CD20、EpCAM、CD24或EGFR，所述第二功能结合片段包含SIRPα胞外D1结构域或其突变体；优选所述第二功能结合片段的氨基酸序列如SEQ ID NO:50所示；更优选所述A链的氨基酸序列如SEQ ID NO:1所示，所述B链的氨基酸序列如SEQ ID NO:2或3所示；所述A链的氨基酸序列如SEQ ID NO:61所示，所述B链的氨基酸序列如SEQ ID NO:62所示；所述A链的氨基酸序列如SEQ ID NO:27所示，所述B链的氨基酸序列如SEQ ID NO:28所示；所述A链的氨基酸序列如SEQ ID NO:29所示，所述B链的氨基酸序列如SEQ ID NO:30所示；所述A链的氨基酸序列如SEQ ID NO:56所示，所述B链的氨基酸序列如SEQ ID NO:57所示。
在一些实施例中，所述第一功能结合片段靶向TIGIT、CD80或PD-1，所述第二功能结合片段包含IL-12A、IL-10单体突变体、IL15与IL-15RαSUSHI复合体，其氨基酸序列分别如SEQ ID NO:51、52、53～54所示；优选所述A链的氨基酸序列如SEQ ID NO:35所示，所述B链的氨基酸序列如SEQ ID NO:36所示；所述A链的氨基酸序列如SEQ ID NO:37所示，所述B链的氨基酸序列如SEQ ID NO:38所示；所述A链的氨基酸序列如SEQ ID NO:39所示，所述B链的氨基酸序列如SEQ ID NO:40或41所示。
在一些实施例中，所述第一功能结合片段靶向CD38或AFP，所述第二功能结合片段包含SIRPα胞外D1结构域或其突变体、或者IFN-β或其突变体；优选所述第二功能结合片段的氨基酸序列如SEQ ID NO:50或55所示；更优选所述A链的氨基酸序列如SEQ ID NO:31所示，所述B链的氨基酸序列如SEQ ID NO:32所示；所述A链的氨基酸序列如SEQ ID NO:33所示，所述B链的氨基酸序列如SEQ ID NO:34所示；或，所述A链的氨基酸序列如SEQ ID NO:58所示，所述B链的氨基酸序列如SEQ ID NO:60所示。
在另一方面，本发明提供编码所述的双特异性重组蛋白的核酸分子；其中编码所述第一功能结合片段的核酸分子与编码第二功能结合片段的核酸在同一条DNA链中，或编码所述第一功能结合片段的核酸分子与编码第二功能结合片段的核酸在不同的DNA链中。
较佳地，编码所述Fc区的核酸分子与编码所述第一功能结合片段或第二功能结合片段的核酸在同一条DNA链中，编码所述Fc区的核酸分子与编码所述第一功能结合片段或第二功能结合片段的核酸在不同的DNA链中。
在另一方面，本发明提供包含上述核酸分子的表达载体。
在另一方面，本发明提供由上述表达载体转化而成的宿主细胞。
在另一方面，本发明还提供该双特异性重组蛋白的制备方法，其使用上述表达载体转化成上述宿主细胞，在适合表达的条件下培养上述宿主细胞，表达即得所述双特异性重组蛋白。
在另一方面，本发明提供所述的双特异性重组蛋白在制备治疗肿瘤、自身免疫性疾病、感染疾病、败血症、移植物抗宿主病、代谢紊乱、内分泌紊乱的药物中的应用。
在一个实施方案中，所述肿瘤为实体瘤或血液瘤；优选的，所述实体瘤选自乳腺癌、结直肠癌、肺癌、胰腺癌、食管癌、子宫内膜癌、卵巢癌、胃癌、前列腺癌、肾癌、宫颈癌、甲状腺癌、子宫癌、膀胱癌、神经内分泌癌、头颈癌、肝癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、皮肤纤维肉瘤突出症、梅克尔细胞癌、胶质母细胞瘤、神经胶质瘤、肉瘤、间皮瘤或骨髓发育不良综合征等；所述血液瘤选自骨髓瘤、淋巴瘤或白血病；优选的，所述自身免疫性疾病选自以下任意一种：桥本氏甲状腺炎、1型糖尿病、系统性红斑狼疮、类风湿性关节炎、干燥综合征等；优选的，所述感染疾病选自以下任意一种：病毒性感染、细菌感染、真菌感染、其他病原体感染等。
在又一个方面，本发明还提供一种药物或药物组合物，其包含本发明的双特异性重组蛋白以及任选的佐剂、赋形剂或药学上可接受的载体。所述药物组合物可以含有药学上可接受的载体。所述组合物可以以任意形式的药物制剂存在，包括但不限于注射剂、粉剂、冷冻干燥粉末等。该药物制剂形式的药物组合物，可以按照制剂学常规技术制备，包括将药物活性成分、本发明的双特异性重组蛋白或融合蛋白与药物载体融合，按照制剂学常规技术制成所需要的剂型。
在一个实施方案中，本发明还提供一种药物组合物，包含编码本发明双特异性重组蛋白的核酸分子的表达载体和任选的可药用的载体。
在另一个方面，本发明还提供一种治疗肿瘤的方法，包括向患者或受试者施用治疗有效量的本发明所述药物组合物。所述肿瘤表达额外的靶标分子，所述靶标包括但不限于5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD71、CD74、CD79b、CD80、CD83、CD86、CD98、CD206、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CCR5、CXCR4、DLL-4、EGFR、EGFRvIII、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、Notch2、Notch1、PD-L1、PD-L2、PD-1、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、TIGIT、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、BCMA、CD138、TROP2、Siglec15、CD155或AFP。
在又一个方面，本发明还提供一种体内基因疗法，包括向患者或受试者引入治疗有效量的编码本发明重组蛋白或融合蛋白的核酸分子或其衍生物。
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。
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。
如本文所用，术语“抗原结合片段”或“fab段”或“fab”由轻链的可变区(VL)、轻链的恒定区(CL)、重链的可变区(VH)、重链的恒定区1(CH1)结构域组成，可与抗原结合。当提及所述抗原结合片段中可变区和恒定区直接连接或通过接头序列Linker连接时，所述恒定区指轻链的恒定区(CL)或重链的恒定区1(CH1)。
如本文所用，术语“第一功能抗原”或“目的抗原”指与第一功能结合片段结合的抗原。
如本文所用，术语“第二功能抗原”指与第二功能结合片段结合的蛋白。
如本文所用，术语“Fc区”(fragment crystallizable，Fc)由IgG恒定区CH2和CH3结构域及铰链区组成。
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'engineeringofantibody 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)。
如本文所用，术语“突变体”，指具有与野生型功能蛋白不同氨基酸序列的功能蛋白或功能片段，例如对功能蛋白或功能片段进行1个或多个氨基酸插入、缺失或取代所形成的新的功能蛋白或功能片段。本文双特异性重组蛋白中所述第二功能结合片段的突变体，特指与对应第二功能抗原的结合亲和力不高于该双特异性重组蛋白第一功能结合片段与第一功能抗原结合亲和力的突变体。
如本文所用，术语“取代”在用于氨基酸残基时是指在肽、多肽或蛋白中将天然存在的或引入的一个或多个氨基酸用另一个取代，形成新的肽、多肽或蛋白。多肽或蛋白中的取代可导致多肽或蛋白功能的增强、减弱或不变。取代还可以是“保守取代”，其针对氨基酸序列时是指将一个氨基酸残基用另一个具有相似理化性质的侧链的不同氨基酸残基取代，或者对那些对多肽的活性并非至关重要的氨基酸的取代。例如，可以在非极性侧链氨基酸残基间(例如Met、Ala、Val、Leu和Ile、Pro、Phe、Trp)、不带电的极性侧链残基间(例如Cys、Ser、Thr、Asn、Gly和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)、β分支侧链氨基酸间(例如Thr、Val和Ile)、含硫侧链氨基酸间(例如Cys和Met)或芳香侧链残基间(例如Trp、Tyr、His和Phe)进行保守取代。在某些实施方式中，取代、删除或添加也可以认为是“保守取代”。插入或删除的氨基酸的数量的范围可以为约1至5。保守取代通常不引起蛋白构象结构上的显著变化，并且因此能保持蛋白的生物学活性。
如本文所用，术语“双阳表达细胞”或“目标细胞”或“目标靶细胞”指可以同时与第一功能结合片段和第二功能结合片段作用的细胞。
如本文所用，术语“第二功能抗原单阳细胞”或“非目标细胞”或“非目标靶细胞”指不与第一功能结合片段作用、只与第二功能结合片段作用的细胞。
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。
如本文所用，术语“D1”、“D2”、“D3”分别指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 cisDimerization ofSignal Regulatory Proteinα(SIRPα)in Binding to CD47.J BiolChem，2010，285(49)：37953-37963)。
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。
如本文所用，术语“IFNα”或“IFN-α”，即“α型干扰素(Interferon-α)”或“干扰素α”，包括所有天然或重组的α型干扰素，是I型干扰素的成员之一，包括IFN-α1a、IFN-α1b、IFN-α2a、IFN-α2b、IFN-α4a、IFN-α4b、IFN-α5、IFN-α6IFN-α7、IFN-α8、IFN-α10、IFN-α14、IFN-α16、IFN-α17和IFN-α21等13个亚型。在本发明中，术语“IFNα”还包括任何具有IFNα生物学活性的物质，例如突变或修饰过的IFNα，例如IFNα的PEG衍生物(PEG-IFNα)。在本发明中，术语“IFNα”不受任何特定的获得来源的限制，可通过市售来源获得或通过本领域技术人员已知的常规技术产生，所述生产方法包括但不限于生物来源提取法和基因工程提取法，其详细描述于例如Pestka S.Arch Biochem Biophys.1983Feb 15；221(1):1-37。在一些实施方案中，IFNα来自选自人、马、牛、鼠、猪、兔、猫、狗、大鼠、山羊、绵羊和非人灵长类动物的物种。特别优选地为人α型干扰素。
如本文所用，术语“IFNα2b”或“IFN-α2b”或“IFNα2b”或“干扰素α2b”或“干扰素-α2b”是IFN-α的一种亚型，均是对干扰素-α2b的表述。
如本文所用，术语“IFN-γ”即γ-干扰素(Interferon-γ)是水溶性二聚体细胞因子，是II型干扰素的唯一成员，由六个α螺旋组成一个核心和在C端区延伸展开的片断序列，常以两个反平行相互锁定的单体形成同二聚体发挥生物活性。
如本文所用，“IFN-β”或“IFNβ”即β-干扰素(β干扰素，Interferon-β)属于人成纤维细胞干扰素，也是Ⅰ型干扰素之一，由成纤维细胞等多种细胞在病毒、核酸等诱导后产生，与IFNα结合相同的干扰素受体，具有相似生物学作用。
如本文所用，术语“IFN-γ融合蛋白”指包含IFN-γ截短体或IFN-γ突变体、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。
如本文所用，术语“IL-10”或“IL10”指白介素10，“IL-10M”或“IL10M”指无需形成同源二聚体即可激活下游信号通路的IL10单体突变体(Josephson,K.Design andanalysis of an engineered human interleukin-10monomer.[J].Journal ofBiological Chemistry,2000,275(18):13552-7.)。
如本文所用，术语“IL-12”或“IL12”指白介素12，由α链(p35亚基，IL-12p35)和β链(p40亚基，IL-12p40)组成的异二聚体分子，所述链通过二硫键共价连接以形成生物学上活性的74kDa异二聚体。如本文所用，术语“IL12A”或“IL-12A”指IL-12的α链。
如本文所用，术语“IL-15”或“IL15”指白介素15，是NK、NKT细胞和CD8记忆T淋巴细胞活性的关键细胞因子，它通过由被称作α、β和γ的三个亚基组成的受体发挥作用，其中α亚基为IL15受体独有。如本文所用，术语“IL-15RαSUSHI”或“IL15RαSUSHI”指IL-15受体α链上的SUSHI结构域，其具有本领域一般含义。所述SUSHI结构域影响IL15激活下游信号通路。
如本文所用，术语“IL-15和IL15RαSUSHI形成的复合体”包括IL15与IL-15RαSUSHI形成的融合蛋白，或IL15与IL-15RαSUSHI根据各种物理或化学方式结合例如离子键、共价键、范德华力等形成的蛋白组合。
如本文所用，术语“接头序列”或“Linker”是指连接不同功能结合片段(如第一功能结合片段和第二功能结合片段、第一功能结合片段或第二功能结合片段和Fc区)，或连接同一功能结合片段内不同结构域的氨基酸序列。
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。
如本文所用，术语“GC33”、“Codrituzumab”在本发明中可以互换使用，表示抗GPC3抗体Codrituzumab。
如本文所用，术语“阿特珠单抗”、“Atezolizumab”在本发明中可以互换使用，表示抗PD-L1抗体Atezolizumab。
如本文所用，术语“宿主细胞”通常包括可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包含本申请公开的多核苷酸，或表达本申请的蛋白质异二聚体(例如，异二聚体蛋白)。宿主细胞可以包括单个宿主细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始亲本细胞完全相同(在形态上或在基因组总DNA互补体上)。宿主细胞可包括用本申请公开的载体在体外转染的细胞。宿主细胞可以是细菌细胞(例如大肠杆菌(E.coli))、酵母细胞或其它真核细胞，例如HEK293细胞、COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞或骨髓瘤细胞。在一些实施方案中，宿主细胞是哺乳动物细胞。在一些实施方案中，所述哺乳动物细胞是CHO细胞。
如在本文中所用，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移至宿主细胞中和/或在宿主细胞之间转移。该术语可包括主要用于将DNA或RNA插入细胞的载体，主要用于DNA或RNA的复制的载体，以及用于DNA或RNA的转录和/或翻译的表达载体。还包括提供不止一种上述功能的载体。“表达载体”是当被引入合适的宿主细胞时可被转录并翻译成多肽的多核苷酸。“表达系统”通常意味着合适的宿主细胞，其包含能够产生期望的表达产量的表达载体。
如本文中所用，术语“细胞增殖”或“增殖”通常是指细胞数目由于分裂而改变的现象。例如，细胞增殖可导致细胞数目的增加。该术语还包括细胞形态通过其已发生改变(例如，尺寸增加)的细胞生长，其与增殖信号一致。
如本文中所用，术语“增殖抑制”或“抑制细胞增殖”通常是指癌细胞的生长速率和/或增殖速率的降低。例如，这可包括癌细胞的死亡(例如通过凋亡)。在一些实施方案中，该术语还可指抑制实体瘤的生长和/或增殖和/或诱导肿瘤的尺寸减小或消除。
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。
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。
与现有技术相比，本发明具有如下有益效果：
本发明通过将双特异性重组蛋白靶向目的抗原的第一功能结合片段的抗原结合片段(fab段)中CL结构域的C末端或CH1结构域的C末端与靶向免疫检查点或免疫检查点配体或细胞因子受体的第二功能结合片段直接连接或通过接头序列连接，同时第二功能结合片段的C端与Fc区的N端直接连接或通过接头序列连接，减弱在制备双特异性重组蛋白时产生可靶向非目标靶细胞免疫检查点、或免疫检查点配体、或细胞因子受体的第二功能结合片段单体或同源二聚体或同源多聚体结构的杂质，意外地降低发酵产物中产品相关重组蛋白混合物杂质(潜在风险杂质)带来的潜在相关安全性风险，提高重组蛋白制备的便利性。
本发明通过将双特异性重组蛋白靶向目的抗原的第一功能结合片段的抗原结合片段(fab段)中CL结构域的C末端或CH1结构域的C末端与靶向免疫检查点或免疫检查点配体或细胞因子受体的第二功能结合片段直接连接或通过接头序列连接，同时第二功能结合片段的C端与Fc区的N端直接连接或通过接头序列连接，出乎意料地平衡和协调两个靶点的安全性和有效性，通过空间构象限制等机理，显著降低双特异性重组蛋白与免疫检查点、或免疫检查点配体、或细胞因子受体单阳高表达的非目标靶细胞或细胞因子受体(第二功能抗原)的结合，提高双特异性重组蛋白的靶向性，降低常见对称或非对称的双抗结构非目标靶细胞靶向结合带来的潜在细胞因子风暴等免疫相关毒副作用，提高药物的安全性。出乎意料地，在维持双特异性重组蛋白第一功能结合片段在目标靶细胞(双阳表达细胞)起效或优效的同时，双特异性重组蛋白相较于含有第二功能结合片段单体/同源二聚体/同源多聚体，其与第二功能抗原单阳细胞(非目标靶细胞)的结合显著下降甚至不结合，但同时，双特异性重组蛋白与双阳表达细胞(即目标靶细胞)上第二功能抗原的相互作用则意外地不弱于、甚至显著强于第二功能结合片段单体/同源二聚体/多聚体，即本发明双特异性重组蛋白技术可显著降低第二功能结合片段与非目标靶细胞第二功能抗原结合而产生相关毒副作用的同时，强化第二功能结合片段与目标靶细胞第二功能抗原结合的作用，显著提升第二功能结合片段对目标细胞的功效。例如，当第二功能结合片段选用SIRPα胞外截短体或非高亲和突变体时，本发明双特异性重组蛋白相比于SIRPα-Fc融合蛋白(如TTI-621)可显著降低其与红细胞或血小板等CD47单阳非目标靶细胞的作用，但同时，本发明双特异性重组蛋白相比于SIRPα-Fc融合蛋白(如TTI-621)可显著提高与双阳表达细胞(目标靶细胞)的相互作用，显著提高免疫细胞(如巨噬细胞)对目标细胞的杀伤作用，同时在目标靶细胞的竞争结合能力显著强于SIRPα-Fc融合蛋白。
此外，本发明的双特异性重组蛋白还能提高部分具有免疫调节功能的融合蛋白的冻融稳定性，例如，当第二功能片段为IFN-α2b或其功能截短体或突变体时，反复冻融5次后，纯度均在95％以上，外观澄清，冻融稳定性明显优于IFN-α2b单体或者PEG化的IFN-α2b。
本发明双特异性重组蛋白结构可拓展性强，筛选设计简单，其第一功能结合片段可选用各抗体序列，第二功能结合片段可选用细胞因子、免疫检查点或免疫检查点配体蛋白及其截短体或突变体，显著降低常规抗体药物筛选所耗费的时间，提高药物筛选效率，降低筛选成本。可选地，第二功能结合片段可选用细胞因子和/或免疫检查点结合蛋白和/或免疫检查点配体结合蛋白或其截短体；优选地，第二功能结合片段可选用内源性细胞因子和/或免疫检查点结合蛋白和/或免疫检查点配体结合蛋白或其截短体，降低潜在的免疫原性。
附图说明
图1是本发明双特异性重组蛋白的结构示意图。
图2是本发明实施例2中双特异性重组蛋白经蛋白A纯化后的SDS-PAGE电泳图。
图3是本发明实施例2中双特异性重组蛋白经亲和捕获后的非还原SDS-PAGE电泳图。
图4是本发明实施例2中双特异性重组蛋白经亲和捕获后的还原SDS-PAGE电泳图。
图5是本发明实施例2中双特异性重组蛋白经精纯后的非还原SDS-PAGE电泳图。
图6是本发明实施例3中流式细胞术测定本发明双特异性重组蛋白与CD20单阳性细胞(非目标靶细胞，CHO-K1-hCD20)的结合曲线图。
图7A是本发明实施例3中流式细胞术测定本发明第二功能抗原为CD47的双特异性重组蛋白及对照样品与CD47单阳细胞(非目标靶细胞，HEK293细胞)的结合曲线图。
图7B是本发明实施例3中流式细胞术测定本发明第二功能抗原为CD47的双特异性重组蛋白及对照样品与CD47单阳细胞(非目标靶细胞，CHO-K1细胞)的结合曲线图。
图8是本发明实施例3中流式细胞术测定本发明第一功能抗原为CD20且第二功能抗原为CD47的双特异性重组蛋白及对照样品与CD20/CD47双阳性细胞(目标靶细胞，Raji细胞)的结合曲线图。
图9A是本发明实施例3中流式细胞术测定本发明第一功能抗原为CD20且第二功能抗原为CD47的双特异性重组蛋白、对应的潜在风险杂质蛋白及对照样品在CD20/CD47双阳细胞(目标靶细胞，Raji细胞)的竞争性结合曲线图。
图9B是本发明实施例3中流式细胞术测定本发明第一功能抗原为EpCAM且第二功能抗原为CD47的双特异性重组蛋白与对照样品在EpCAM/CD47双阳细胞(目标靶细胞，CAPAN-2细胞)的竞争性结合曲线图。
图9C是本发明实施例3中流式细胞术测定本发明第一功能抗原为CD24且第二功能抗原为CD47的双特异性重组蛋白与对照样品在CD24/CD47双阳细胞(目标靶细胞，MCF-7细胞)的竞争性结合曲线图。
图9D是本发明实施例3中流式细胞术测定本发明第一功能抗原为CD38且第二功能抗原为CD47的双特异性重组蛋白与对照样品在CD38/CD47双阳细胞(目标靶细胞，Raji细胞)的竞争性结合曲线图。
图10是流式细胞术测定本发明实施例4中第一功能抗原为GPC3且第二功能结合蛋白为IFN-α2b的双特异性重组蛋白与对照样品在目标靶细胞HepG2肝癌细胞的结合活性曲线图。
图11是流式细胞术测定本发明实施例4中第一功能抗原为GPC3且第二功能结合蛋白为IFN-α2b的双特异性重组蛋白与对照样品在目标靶细胞HuH-7的结合活性柱状图。
图12是LDH方法测定本发明实施例5中双特异性重组蛋白在目标靶细胞HepG2的ADCC活性曲线图。
图13是本发明实施例6中不同接头序列的双特异性重组蛋白对目标靶细胞HuH-7增殖抑制活性曲线图。
图14是本发明实施例6中具有GPC3抗原靶向功能的双特异性重组蛋白与对照样品对GPC3阳性的目标靶细胞HuH-7增殖抑制活性曲线图。
图15是本发明实施例7中双特异性重组蛋白对PD-L1阳性的目标靶细胞MDA-MB-231的增殖抑制活性曲线图。
图16是本发明实施例7中双特异性重组蛋白对抗PD-L1抗体封闭后的MDA-MB-231的增殖抑制活性曲线图。
图17A是本发明实施例8中双特异性重组蛋白对CD38阳性的目标靶细胞Daudi增殖抑制活性曲线图。
图17B是本发明实施例8中双特异性重组蛋白对CD38阴性的非目标靶细胞SK-BR3的增殖抑制活性曲线图。
图18是本发明实施例9中含不同IFN-α2b低亲和突变体的双特异性重组蛋白对GPC3阳性的目标靶细胞HuH-7增殖抑制活性曲线图。
图19是本发明实施例9中含不同IFN-α2b低亲和突变体的双特异性重组蛋白对GPC3阴性的非目标靶细胞SW480增殖抑制活性曲线图。
图20是本发明实施例9中含不同IFN-α2b低亲和突变体的双特异性重组蛋白对GPC3阴性的非目标靶细胞U266增殖抑制活性曲线图。
图21是本发明实施例10中双特异性重组蛋白潜在风险杂质对GPC3阴性的非目标靶细胞MDA-MB-231增殖抑制活性曲线图。
图22是本发明实施例11中双特异性重组蛋白对抗TIGIT抗体封闭后的TIGIT阳性目标靶细胞H_IL12 Reporter 293的结合活性曲线图。
图23是本发明实施例12中双特异性重组蛋白对THP1细胞P-STAT3激活水平检测结果。
图24是本发明实施例13中双特异性重组蛋白对OKT3刺激48小时后PD-1阳性的hPBMC的增殖活性检测结果。
图25是本发明实施例13中双特异性重组蛋白对PD-1阴性的非目标靶细胞M-07e的增殖活性检测结果。
具体实施方式
实施例1表达载体的构建
双特异性重组蛋白由GENEWIZ公司根据蛋白序列进行密码子优化后直接合成，联入pCDNA3.1质粒中，经测序确定。将上述不同的表达质粒混合配对转染表达细胞，获得双特异性重组蛋白或对照样品(见表1)。后续的实验材料均由此系列质粒转染表达细胞后，提取获得。
表1双特异性重组蛋白及对照样品示例性分子结构
Figure BDA0003268638110000241
Figure BDA0003268638110000251
Figure BDA0003268638110000261
Figure BDA0003268638110000271
Figure BDA0003268638110000281
以上表1中，双特异性重组蛋白的第一功能结合片段通过其靶向的靶标抗原来表征，即第一功能抗原；(H)指重链VH和CH1组成结构域，(L)指轻链VL和CL组成结构域；D1表示人源SIRPα野生型及其突变体的胞外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。所述序列名称对应序列编号见表2。其中，信号肽的序列如SEQ ID NO:49所示。Codrituzumab的氨基酸序列引用自专利US7919086，重链氨基酸序列如SEQ ID NO:19所示，轻链氨基酸序列如SEQ ID NO:20所示。Atezolizumab的氨基酸序列引用自专利US20100203056，重链氨基酸序列如SEQ ID NO:21所示，轻链氨基酸序列如SEQ ID NO:22所示。Tiragolumab的氨基酸序列重链氨基酸序列如SEQ ID NO:61所示，轻链氨基酸序列如SEQ ID NO:62所示。Palivizumab重链可变区和轻链可变区的氨基酸序列引用自专利WO1994017105。人IgG1同种型对照(B117901)购自百英生物。重组表达IFN-α2b蛋白(Z03003)购买自南京金斯瑞生物科技有限公司。IL10M-Fc中IL10采用单体突变体形式，对应的序列参考下述文献(Josephson,K.Design and analysis ofan engineered human interleukin-10monomer.[J].Journal of BiologicalChemistry,2000,275(18):13552-7.)。IL15-IL15RαSUSHI-Fc(C15Y)购自近岸蛋白质(novoprotein)。
表2序列名称与对应序列编号
Figure BDA0003268638110000282
Figure BDA0003268638110000291
Figure BDA0003268638110000301
Figure BDA0003268638110000311
表1中，IFNα2b采用的是人IFN-α2b(Genebank：AAP20099.1)，以此为例来阐述本发明双特异性重组蛋白中第二功能结合片段为IFNα的设计。IFNα家族共15个亚型，不同亚型结构相似，序列高度同源(80-99％)，且所结合的IFN受体一致，都具有抗病毒、增殖抑制、抗肿瘤和免疫调节的功能。对于IFN-α2b可以得到的技术效果，IFN-α其余亚型可以达到一致的技术效果(British Journal of Pharmacology(2013)168 1048–1058)，本发明在这里不再赘述。IFN-β和IFN-α2b具有相同的干扰素受体，且同为I型干扰素，具有相似生物学作用，因此亦可达到一致的技术效果。
本发明双特异性重组蛋白的结构如图1所示，所述双特异性重组蛋白靶向目的抗原的第一功能结合片段包含抗原结合片段，其中抗原结合片段的CL结构域的C末端或CH1结构域的C末端与靶向免疫检查点或免疫检查点配体或细胞因子受体的第二功能结合片段直接连接或通过接头序列连接。第一功能结合片段的抗原结合片段中可变区(V区)和恒定区(C区)直接连接或通过接头序列连接；或所述抗原结合片段与Fc直接连接或通过接头序列连接；或同时使用上述两种方式连接。第一功能结合片段的轻链VL或重链VH与第二功能结合片段是通过knobs-into-holes结合的。所述第一功能结合片段的轻链VL或重链VH或第二功能结合片段与所述Fc区直接连接或通过接头序列连接。例如，图1中，a、e代表第一功能结合片段的抗原结合片段的CL结构域的C末端与第二功能结合片段直接或通过接头序列连接；b、f代表第一功能结合片段的抗原结合片段的CH1结构域的C末端与第二功能结合片段直接或通过接头序列连接；c、g、j、k代表第一功能结合片段的抗原结合片段的CL结构域的C末端与第二功能结合片段直接或通过接头序列连接，且Fc区通过knobs-into-holes结合；d、h、i、l代表第一功能结合片段的抗原结合片段的CH1结构域的C末端与第二功能结合片段直接或通过接头序列连接，且Fc区通过knobs-into-holes结合。
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
1、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。
2、细胞转染、蛋白表达
以下方法以LCB-001为例，适用于第二功能抗原是CD47的双特异性重组蛋白。
将所得表达质粒用0.22μm滤膜过滤后，吸取3mg质粒(其中，双特异性重组蛋白，其A链和B链表达质粒的比例为1:1(摩尔比))加入到50mL Opti MEM I Reduced SerumMedium(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 SerumMedium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(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80g CD Efficient FeedC AGT(Gibco)、75g 5×00483(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。
以下方法以LCB-009为例，适用于第二功能抗原除CD47外的双特异性重组蛋白。
将所得表达质粒用0.22μm滤膜过滤后，吸取50μg质粒(其中，A链和B链表达质粒的质量比例为2:1或者3:1)加入到2mL OptiPRO SFM Medium(GIBCO)中混匀。吸取160μL转染试剂ExpiFectamine CHO Reagent加入到2mL OptiPRO SFM Medium中混匀。将得到的转染试剂混合溶液加入到含有质粒的混合溶液中混匀。将质粒和转染试剂的混合物缓慢均匀加入体积为50mL，细胞密度为6×106活细胞/mL的宿主细胞ExpiCHO-S(Thermo Fisher)悬液中，置于37℃、8％CO2培养箱中培养。第1天(18-22小时后)加入300μL ExpiCHO Enhancer和8mL ExpiCHO Feed，将培养温度降低至32℃；第5天进行第二次补料，补加8mL ExpiCHOFeed，培养12天后收获。将细胞悬液8000rpm离心15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。
3、蛋白纯化
1)样品捕获(蛋白A亲和捕获)
以下方法以LCB-001为例，该方法适用于所有本发明双特异性重组蛋白。
将LCB-001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(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。
2)样品精纯
以下方法以LCB-009为例阐述双特异性重组蛋白精纯的步骤，适用于蛋白A亲和捕获后聚体含量较多的双特异性重组蛋白。
双特异性重组蛋白LCB-009表达上清使用SULFATE 650F填料(TOSOH)除去样品中的聚体及其它杂质，实验过程如下：
a)平衡：使用平衡液(50mM NaAC-HAC，pH5.5)平衡层析柱，直至紫外检测线平稳；
b)上样：用样品泵上样，保留时间5min，载量≤50mg/mL；
c)再平衡：使用平衡液(50mM NaAC-HAC，pH5.5)冲洗层析柱5个柱体积；
d)洗脱：使用洗脱液(50mM NaAC-HAC，250Mm，pH5.5)洗脱目的蛋白，SDS-PAGE检测蛋白纯度。
表1中对照样品的纯化步骤参照1)样品捕获(蛋白A亲和捕获)的操作步骤获得。
纯化后双特异性重组蛋白、对照样品及潜在风险杂质的SDS-PAGE蛋白电泳检测结果如图2-图5所示。
LCB-001、LCB-002、LCB-001-R、LCB-002-R四种蛋白的理论分子量分别为：111kD、114kD、123kD和129kD。如图2所示，各泳道的目标蛋白均表达正常，但LCB-001和LCB-002(图2泳道1和泳道2)可见不同程度的左臂二聚体、右臂二聚体(LCB-001-R、LCB-002-R)和/或多聚体。
LCB-009、LCB-010、LCB-011、LCB-012、LCB-013、LCB-014、LCB-015、LCB-010-M1、LCB-010-M2、LCB-010-M3、LCB-010-M4、LCB-010-M5、LCB-011-M3、LCB-011-M4的理论分子量均为120kD左右，表1所示对照抗体Codrituzumab分子量为150kD，IFNα2b-Fc的分子量为88kD，IFNα2b单体的分子量为19.2kD。亲和捕获(即经蛋白A纯化后)的样品非还原SDS-PAGE蛋白电泳检测结果如图3所示，还原SDS-PAGE蛋白电泳检测结果如图4所示，电泳结果显示该结构表达抗体聚体含量较多，精纯后(即经SULFATE 650F纯化后)的非还原SDS-PAGE蛋白电泳检测结果如图5所示，可见通过阳离子交换层析填料可去除大部分聚体。
其他表1所示双特异性重组蛋白通过亲和捕获及精纯后，样品在还原及非还原的SDS-PAGE蛋白电泳检测结果与理论分子量均一致，在此不再赘述。
实施例3第二功能抗原为CD47的双特异性重组蛋白靶标亲和力、靶标竞争结合活性检测
1、CD47和/或CD20靶标亲和力检测方法
流式细胞术检测双特异性重组蛋白对靶标CD20的亲和力：
双特异性重组蛋白对靶标CD20的结合亲和力通过流式细胞术进行测定。以下方法以LCB-001或LCB-002为例，适用于第一功能抗原为CD20的重组蛋白的检测。
培养CHO-K1-hCD20(过表达hCD20的中国仓鼠卵巢上皮细胞)，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8个稀释度的LCB-001、LCB-002、阳性对照Ofatumumab、阳性对照Rituximab或阴性对照IgG(Isotype)(从100nM起始，3倍梯度稀释，共8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(BD)检测荧光值。
由于CHO-K1细胞不表达CD47抗原，因此可以在细胞水平上用CHO-K1-hCD20细胞评价重组蛋白LCB-001、LCB-002，阳性对照样品Ofatumumab、Rituximab，阴性对照IgG与CD20的结合亲和力。
试验结果显示，除阴性对照IgG不能结合CHO-K1外，重组蛋白LCB-001、LCB-002，阳性对照样品Ofatumumab、Rituximab均能与CHO-K1细胞结合；LCB-001、LCB-002对CD20的结合亲和力与抗CD20抗体Ofatumumab或Rituximab的结合亲和力相似。
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CD20抗原，且与CD20的结合亲和力不低于同靶点单克隆抗体对CD20的结合亲和力；本发明的重组蛋白能够高亲和靶向目标靶细胞。
例如，如图6所示，重组蛋白LCB-001、LCB-002，阳性对照样品Ofatumumab、Rituximab均能与CHO-K1-hCD20细胞结合。具体地，LCB-001、LCB-002对CD20的结合亲和力与抗CD20抗体Ofatumumab或Rituximab的结合亲和力相似。
流式细胞术检测双特异性重组蛋白对靶标CD47的亲和力：
双特异性重组蛋白对靶标CD47的结合亲和力通过流式细胞术进行测定。以下方法以LCB-001、LCB-002或LCB-017为例，适用于第二功能抗原为CD47的重组蛋白的检测。
培养HEK293(人胚胎肾细胞293)(CD20-/CD47+，非目标靶细胞)，收集生长良好的细胞并计数，离心并用PBS+2％FBS(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7个稀释度的LCB-001、LCB-002、抗CD47抗体Magrolimab、抗CD47融合蛋白TTI-621或IgG1(Isotype)(从100nM起始，4倍梯度稀释，共7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(BD)检测荧光值。
由于HEK293细胞不表达CD20抗原，因此可以在细胞水平上用HEK293细胞评价双特异性重组蛋白LCB-001、LCB-002，阳性对照抗CD47抗体Magrolimab、抗CD47融合蛋白TTI-621，及阴性对照IgG与CD47的结合亲和力。
试验结果显示，除阴性对照IgG不能结合HEK293外，抗CD47抗体Magrolimab、抗CD47融合蛋白TTI-621(SIRPαD1-Fc融合蛋白)、重组蛋白LCB-001和LCB-002均能与HEK293细胞结合；重组蛋白LCB-001和LCB-002与HEK293的结合显著弱于抗CD47抗体Magrolimab，同时亦非显而易见地显著弱于抗CD47融合蛋白TTI-621。
例如，如图7A所示，抗CD47抗体Magrolimab、抗CD47融合蛋白TTI-621、重组蛋白LCB-001和LCB-002均能与HEK293细胞结合。具体地，重组蛋白LCB-001和LCB-002的亲和力极显著弱于抗CD47抗体Magrolimab，显著弱于抗CD47融合蛋白TTI-621(SIRPαD1-Fc融合蛋白)。
CD47转染的CHO-K1(中国仓鼠卵巢细胞)(CD38-/CD47+，非目标靶细胞)，洗涤消化后，收集生长良好的细胞并计数，离心并用DPBS+2％FBS(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7个稀释度的LCB-017、Felzartamab、TTI-621、或SIRPα-D1m-Fc(从200nM起始，3倍梯度稀释，共11个浓度点)，并将96孔板在冰箱中4℃孵育1小时；用DPBS+2％FBS清洗后，加入山羊抗人IgG Fc-FITC(F9512-2ML，Sigma)4℃孵育1小时；用DPBS+2％FBS清洗重悬后，通过流式细胞仪(BD)检测荧光值。
由于CD47转染的CHO-K1细胞不表达CD38抗原，因此可以在细胞水平上用该CHO-K1细胞评价双特异性重组蛋白LCB-017、阴性对照抗CD38抗体Felzartamab、阳性对照抗CD47融合蛋白TTI-621及其高亲和SIRPαD1m-Fc与CD47的结合亲和力。
试验结果显示，除阴性对照抗CD38抗体Felzartamab不能结合CHO-K1外，抗CD47融合蛋白TTI-621(SIRPαD1-Fc融合蛋白)、高亲和SIRPαD1m-Fc融合蛋白、重组蛋白LCB-017均能与HEK293细胞结合；重组蛋白LCB-017与非目标靶细胞CHO-K1的结合显著弱于抗CD47融合蛋白TTI-621。
例如，如图7B所示，高亲和SIRPαD1m-Fc融合蛋白、抗CD47融合蛋白TTI-621、重组蛋白LCB-017均能与非目标靶细胞CHO-K1细胞结合。具体地，重组蛋白LCB-017的亲和力极显著弱于抗CD47融合蛋白TTI-621(SIRPαD1-Fc融合蛋白)。
其他第二功能抗原为CD47的本发明双特异性重组蛋白对非目标靶细胞同样可以观察到结合亲和力较低或不结合的现象。
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的结合亲和力显著弱于SIRPαD1-Fc融合蛋白对CD47的结合亲和力；本发明的双特异性重组蛋白能够意外地显著降低或避免抗CD47抗体或SIRPαD1-Fc融合蛋白治疗产生的红细胞凝集、贫血、血小板减少症等非肿瘤靶细胞杀伤等副作用。
表面等离子体共振分析(后称“SPR分析”)双特异性重组蛋白对靶标CD20或CD47的 亲和力：
以下方法以LCB-002为例，适用于第一功能抗原为CD20、第二功能抗原为CD47的重组蛋白的检测。
对于SPR分析来说，使用Biacore T200(GE Healthcare)。将抗人IgG(Fc)抗体(人抗体捕获试剂盒，GE Healthcare)固定在CM5传感器芯片上。允许用HBS-EP(GEHealthcare)稀释的5μg/mL的人CD20-Fc或人CD47-Fc嵌合蛋白各自固定，并测量捕获量。随后，将完全的重组蛋白LCB-002、抗CD20抗体Ofatumumab、抗CD47融合蛋白TTI-621用HBS-EP稀释到50nM，并分别测量其与人CD20-Fc嵌合蛋白或人CD47-Fc嵌合蛋白的结合量。接着，以流速50μL/分钟添加HBS-EP+buffer 5分钟，测定完重组蛋白LCB-002、抗CD20抗体Ofatumumab、抗CD47融合蛋白TTI-621和人CD20-Fc嵌合蛋白或人CD47-Fc嵌合蛋白的解离。用Fit local分析二价分析物模型、Rmax，算出结合速度常数(ka)及解离速度常数(kd)，用kd除以ka，由此算出结合解离常数(KD)。重组蛋白LCB-002、抗CD20抗体Ofatumumab、抗CD47融合蛋白TTI-621与人CD20-Fc嵌合蛋白或人CD47-Fc嵌合蛋白的Kd值如表3所示：
表3 SPR分析重组蛋白或对照样品与第一或第二功能抗原的结合解离常数
样品名称 Kd(hCD20) Kd(hCD47)
LCB-002 3.24nM 52.8nM
Ofatumumab 1.98nM -
TTI-621 - 37.3nM
由于Ofatumumab为特异性靶向人CD20蛋白，因此不与人CD47-Fc嵌合蛋白结合，而抗CD47融合蛋白TTI-621则特异性靶向CD47蛋白，因此不与人CD20-Fc嵌合蛋白结合。
试验结果显示，蛋白水平上，重组蛋白LCB-002能分别与人CD47蛋白或人CD20蛋白结合，且其亲和力相比抗CD20抗体Ofatumumab或抗CD47融合蛋白TTI-621未见明显改变。
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CD20抗原和CD47抗原，且与CD47或CD20的结合亲和力与抗CD20抗体Ofatumumab或抗CD47融合蛋白TTI-621相当。
流式细胞术检测与靶标CD20和CD47的双特异性结合情况：
双特异性重组蛋白对靶标CD20和CD47的结合亲和力通过流式细胞术进行测定。以下方法以LCB-001或LCB-002为例，适用于第一功能抗原为CD20、第二功能抗原为CD47的重组蛋白的检测。
Raji细胞(人B细胞淋巴瘤)(上海中科院细胞库)(CD20+/CD47+，目标靶细胞)，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)，分别加入8个稀释度的LCB-001、LCB-002、Ofatumumab、Rituximab、IgG(从100nM起始，3倍梯度稀释，共8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(BD)检测荧光值。
如图8所示，由于Raji细胞表面同时表达CD20和CD47抗原，因此，抗CD20抗体Ofatumumab、Rituximab均能与Raji细胞特异性结合，而重组蛋白LCB-001和LCB-002也能与Raji细胞结合，且结合力与Ofatumumab、Rituximab在相似水平。
2、靶标竞争结合活性检测
以下方法以LCB-001、LCB-005、LCB-006、LCB-017为例，适用于第二功能抗原为CD47的双特异性重组蛋白。
流式细胞术检测LCB-001的竞争结合活性：
双特异性重组蛋白LCB-001、LCB-002及潜在杂质对照样品LCB-001-R、LCB-002-R与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争结合细胞表面CD47的竞争结合活性通过流式细胞术进行测定。以下方法以LCB-001或LCB-002为例，适用于第一功能抗原为CD20、第二功能抗原为CD47的重组蛋白的检测。
Raji细胞(人B细胞淋巴瘤)(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)，将梯度稀释的LCB-001(起始终浓度200000ng/mL，5倍稀释，共8个浓度点)。FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)终浓度为4nM。将LCB-001、SIRPα(CV1)-Fc-FITC和Raji细胞共同孵育1h，离心弃去上清，用DPBS+2％FBS重悬细胞，然后用流式细胞仪检测。
试验结果显示，抗CD47抗体Magrolimab、抗CD47融合蛋白TTI-621、重组蛋白LCB-001和LCB-002均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc竞争性结合Raji细胞上的CD47抗原，发挥竞争结合活性；潜在杂质LCB-001-R、LCB-002-R无法与FITC标记的高亲和SIRPαD1m-Fc竞争性结合CD47抗原，无法发挥竞争结合活性。
例如，如图9A所示，抗CD47抗体Magrolimab、抗CD47融合蛋白TTI-621、重组蛋白LCB-001和LCB-002均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc竞争性结合CD47抗原，发挥竞争结合活性，且重组蛋白LCB-002竞争结合活性非显而易见地显著优于抗CD47融合蛋白TTI-621，重组蛋白LCB-001竞争结合活性与抗CD47融合蛋白TTI-621相当。该结果显示，采用合适接头序列连接第一功能结构片段和第二功能结构片段的重组蛋白可非显而易见地显著提高第二功能结构片段与第二功能抗原的竞争结合能力。同时，潜在风险杂质蛋白LCB-001-R、LCB-002-R均无法与FITC标记的高亲和SIRPαD1m-Fc竞争结合CD47抗原。根据该结果显示，重组蛋白制备过程中产生的难以完全去除的潜在风险杂质对第二功能抗原的竞争结合活性显著降低，可以推论，该潜在风险杂质结合非目标靶细胞上的第二功能抗原非常弱，极大地减弱了免疫相关毒副作用，具有极佳的安全性。
流式细胞术检测LCB-005的竞争结合活性：
双特异性重组蛋白LCB-005与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争结合细胞表面CD47的竞争结合活性通过流式细胞术进行测定。以下方法以LCB-005为例，适用于第一功能抗原为EpCAM、第二功能抗原为CD47的重组蛋白的检测。
将生长良好的目标靶细胞CAPAN-2细胞(人胰腺癌细胞)(EpCAM+/CD47+)(购自南京科佰)，洗涤消化后，计数离心并用DPBS+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)，将LCB-005进行梯度稀释(起始终浓度200nM，3倍稀释，共8个浓度点)。FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)终浓度为4nM，将LCB-006、SIRPαD1m-Fc、TTI-621、SIRPα(CV1)-Fc-FITC和CAPAN-2细胞共同孵育1h，离心弃去上清，用DPBS+2％FBS重悬细胞，然后用流式细胞仪检测。
试验结果显示，相较于在CAPAN-2细胞上未有明显竞争结合活性的抗CD47融合蛋白TTI-621，双特异性重组蛋白LCB-005、高亲和SIRPαD1m-Fc均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争性结合CAPAN-2细胞上的CD47抗原，发挥竞争结合活性，双特异性重组蛋白LCB-005对CAPAN-2的竞争结合活性显著优于抗CD47融合蛋白TTI-621。
例如，如图9B所示，双特异性重组蛋白LCB-005和高亲和SIRPαD1m-Fc均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争性结合CD47抗原，发挥竞争结合活性，而抗CD47融合蛋白TTI-621未见竞争结合活性，可见双特异性重组蛋白LCB-005竞争结合活性非显而易见地显著优于抗CD47融合蛋白TTI-621。该结果显示，本发明双特异性重组蛋白采用合适接头序列连接第一功能结构片段和第二功能结构片段的重组蛋白可非显而易见地显著提高第二功能结构片段与第二功能抗原的竞争结合能力。
流式细胞术检测LCB-006的竞争结合活性：
双特异性重组蛋白LCB-006与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争结合细胞表面CD47的竞争结合活性通过流式细胞术进行测定。以下方法以LCB-006为例，适用于第一功能抗原为CD24、第二功能抗原为CD47的重组蛋白的检测。
将生长良好的目标靶细胞MCF-7细胞(人乳腺癌细胞)(CD24+/CD47+)(购自南京科佰)，洗涤消化后，计数离心并用DPBS+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)，将LCB-006进行梯度稀释(起始终浓度200nM，3倍稀释，共8个浓度点)。FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)终浓度为4nM，将LCB-006、SIRPαD1m-Fc、TTI-621、SIRPα(CV1)-Fc-FITC和MCF-7细胞共同孵育1h，离心弃去上清，用DPBS+2％FBS重悬细胞，然后用流式细胞仪检测。
试验结果显示，相较于在MCF-7细胞上未有明显竞争结合活性的抗CD47融合蛋白TTI-621，双特异性重组蛋白LCB-006、高亲和SIRPαD1m-Fc均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争性结合MCF-7细胞上的CD47抗原，发挥竞争结合活性，双特异性重组蛋白LCB-006对MCF-7的竞争结合活性显著优于抗CD47融合蛋白TTI-621。
例如，如图9C所示，双特异性重组蛋白LCB-006和高亲和SIRPαD1m-Fc均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争性结合CD47抗原，发挥竞争结合活性，而抗CD47融合蛋白TTI-621未见竞争结合活性，可见双特异性重组蛋白LCB-006竞争结合活性非显而易见地显著优于抗CD47融合蛋白TTI-621。该结果显示，本发明双特异性重组蛋白采用合适接头序列连接第一功能结构片段和第二功能结构片段的重组蛋白可非显而易见地显著提高第二功能结构片段与第二功能抗原的竞争结合能力。
流式细胞术检测LCB-017的竞争结合活性：
双特异性重组蛋白LCB-017与FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)竞争结合细胞表面CD47的竞争结合活性通过流式细胞术进行测定。以下方法以LCB-017为例，适用于第一功能抗原为CD38、第二功能抗原为CD47的重组蛋白的检测。
Raji细胞(人B细胞淋巴瘤)(上海中科院细胞库)(CD38+/CD47+)，收集生长良好的细胞计数并离心，并用DPBS+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)，将LCB-017进行梯度稀释(起始终浓度200nM，3倍稀释，共7个浓度点)。FITC标记的高亲和SIRPαD1m-Fc(SIRPα(CV1)-Fc-FITC)终浓度为4nM。将LCB-017、SIRPαD1m-Fc、TTI-621、SIRPα(CV1)-Fc-FITC和Raji细胞共同孵育1h，离心弃去上清，用DPBS+2％FBS重悬细胞，然后用流式细胞仪检测。
试验结果显示，抗CD47融合蛋白TTI-621、双特异性重组蛋白LCB-017和高亲和SIRPαD1m-Fc均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc竞争性结合Raji细胞上的CD47抗原，发挥竞争结合活性，双特异性重组蛋白LCB-017对MCF-7的竞争结合活性显著优于抗CD47融合蛋白TTI-621。
例如，如图9D所示，抗CD47融合蛋白TTI-621、双特异性重组蛋白LCB-017和高亲和SIRPαD1m-Fc均能不同程度地与FITC标记的高亲和SIRPαD1m-Fc竞争性结合Raji细胞上的CD47抗原，发挥竞争结合活性，可见双特异性重组蛋白LCB-017竞争结合活性(IC50＝27.98)非显而易见地显著优于抗CD47融合蛋白TTI-621(IC50＝1067)。该结果显示，本发明双特异性重组蛋白采用合适接头序列连接第一功能结构片段和第二功能结构片段的重组蛋白可非显而易见地显著提高第二功能结构片段与第二功能抗原的竞争结合能力。
综上所述(图9A-图9D)，本发明双特异性重组蛋白采用合适接头序列连接第一功能结构片段和第二功能结构片段的重组蛋白可非显而易见地显著提高第二功能结构片段与第二功能抗原的竞争结合能力。其他第二功能抗原为CD47的双特异性重组蛋白在目的抗原阳性的目标靶细胞上也观察到了与上述数据一致的结果。
实施例4靶向GPC3双特异性重组蛋白对GPC3高表达肝癌细胞系(双阳表达细胞，目标靶细胞)结合活性检测
靶向GPC3双特异性重组蛋白对靶标GPC3高表达肝癌细胞系的结合亲和力通过流式细胞术进行测定。以下方法以LCB-009、LCB-010、LCB-011为例，适用于第一功能结合片段结合GPC3抗原、第二功能结合片段为IFN-α2b或其低亲和突变体的双特异性重组蛋白的检测。
检测细胞为HepG2细胞(南京科佰生物科技有限公司)和HuH-7(上海中科院细胞库)，收集生长良好的细胞并计数，离心并用FACS缓冲液(PBS+2％FBS)重悬细胞至1×106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)，分别加入7个稀释度的双特异性重组蛋白或对照蛋白(从100nM起始，3倍梯度稀释，共7个浓度)，4℃孵育1小时；FACS缓冲液清洗后，加入山羊抗人IgG(Alexa Fluor488 goat anti-human IgG(H+L)，Invitrogen)4℃孵育1小时；用FACS缓冲液清洗重悬后，通过流式细胞仪(Attune Nxt，invitrogen)检测荧光值。
实验结果如图10和图11所示，双特异性重组蛋白LCB-009、LCB-010、LCB-011均可与同时表达GPC3和IFNα受体的目标靶细胞HepG2和HuH-7细胞具有一定的结合活性，双特异性重组蛋白与HepG2和HuH-7细胞的结合相较于抗GPC3单抗(对照样品Codrituzumab)具有更高的最大平均荧光强度。同时，如图10和图11所示，接头序列的长短对双特异性重组蛋白与目标靶细胞的结合活性影响较弱，当接头序列较短(如接头序列为1个GGGGS)时，双特异性重组蛋白(例如LCB-009)与目标靶细胞的结合活性(EC50)相对较低。
实施例5靶向GPC3双特异性重组蛋白的抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity，ADCC)活性检测
双特异性重组蛋白对靶标GPC3高表达肝癌细胞系的ADCC活性通过乳酸脱氢酶(LDH)方法进行测定。以下方法以LCB-009、LCB-010、LCB-011为例，适用于第一功能结合片段结合GPC3抗原、第二功能结合片段为IFN-α2b或其低亲和突变体的双特异性重组蛋白ADCC活性检测。
人外周血单核细胞(Peripheral Blood Mononuclear Cell，PBMC)作为效应细胞，靶细胞为GPC3高表达的HepG2肝癌细胞系，LDH检测试剂盒为CytoTox-ONETM-HomogeneousMembrance Integrity Assay，Promega，G7892。靶细胞和效应细胞以1:20的条件铺板，加入双特异性重组蛋白或对照蛋白(从150nM起始，5倍梯度稀释，共8个浓度)，37℃共孵育4小时。37℃孵育3.5小时后向对照组加入裂解试剂，显微镜下观察细胞状态，待细胞裂解完全后10000转离心5分钟，取上清转至96孔黑底透明板内(Corning,3904)，与反应底物于37℃孵育30分钟，加入终止液，避光振荡3到5分钟，酶标仪(SpectraMax M2)检测信号值，详细步骤参考LDH检测试剂盒说明书。
如图12所示，双特异性重组蛋白(如LCB-009、LCB-010、LCB-011)保留有ADCC活性，且双特异性重组蛋白的ADCC活性与抗GPC3单抗(对照样品Codrituzumab)相当(EC50＝0.53)。
实施例6靶向GPC3双特异性重组蛋白增殖抑制活性检测
靶向GPC3的双特异性重组蛋白对不同肿瘤细胞系的增殖抑制活性通过celltiter glo试剂盒(Promega，Cat:G7558)进行测定。以下方法以LCB-009、LCB-010、LCB-011为例，适用于第一功能结合片段结合GPC3抗原、第二功能结合片段为IFNα2b或其低亲和突变体的双特异性重组蛋白的检测。
GPC3阳性细胞系HuH-7或者GPC3阴性肿瘤细胞系U266(购自南京科佰生物科技有限公司)和GPC3阴性肿瘤细胞系SW480(购自南京科佰生物科技有限公司)铺板于96孔黑底透明板内(Corning，3904)，加入双特异性重组蛋白或对照蛋白(52nM起始，10倍梯度稀释，共6个点；或10nM起始，5倍梯度稀释，共8个点)，放置二氧化碳培养箱37℃培养3天，加入Cell titer glo，Multomode Plate Reader(PerkinElmer，Envision2105)检测信号值。
结果显示，双特异性重组蛋白(如LCB-009、LCB-010、LCB-011)对GPC3阳性(GPC3+)的目标靶细胞HuH-7增殖抑制活性(IC50)均高于对照IFN-α2b(如图13所示)，也高于抗GPC3单抗(anti-GPC3 mAb，即对照样品Codrituzumab，该抗体对HuH-7未见增殖抑制作用)。如图14所示，缺乏GPC3靶向功能的双特异性重组蛋白LCB-012以及IFN-α2b的Fc融合蛋白(IFN-α2b-Fc)对HuH-7(GPC3阳性的目标靶细胞)的增殖抑制活性均显著低于拥有GPC3靶向功能的双特异性重组蛋白LCB-010，表明拥有GPC3靶向功能的双特异性重组蛋白其与目标靶细胞上GPC3的结合，可以显著地增强IFN-α2b增殖抑制活性，而不具有目标靶细胞靶向作用的双特异性重组蛋白其IFN-α2b所起到的增殖抑制活性较低，揭示本发明双特异性重组蛋白仅对具有目的抗原的目标靶细胞有较强的增殖抑制作用，而对于不具有目的抗原的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋白的安全性较高。
另，如表4结果显示，不同接头序列的双特异性重组蛋白(以LCB-009、LCB-010、LCB-011为例)对GPC3阳性(GPC3+)的目标靶细胞(以HuH-7为例)均具有相较IFN-α2b更强的增殖抑制活性，不同接头序列的双特异性重组蛋白其增殖抑制活性略有差异，含有1个GGGGS作为接头序列的双特异性重组蛋白(即以较短长度接头序列连接的双特异性重组蛋白，以(LCB-009为例)活性相对较低(如图13、表4所示)，该结果与图11所示不同接头序列的重组蛋白结合活性结果相一致。但在GPC3阴性(GPC3-)的非目标靶细胞U266和SW480细胞系中，双特异性重组蛋白(以LCB-009、LCB-010、LCB-011为例)的增殖抑制活性显著低于IFN-α2b(即双特异性重组蛋白的相对活性远小于1)，该结果表明在不表达GPC3的细胞(即不表达目的抗原的非目标靶细胞)中，双特异性重组蛋白增殖抑制活性极低，提示其安全性较高。同时，如表4所示，双特异性重组蛋白对目标靶细胞的增殖抑制活性是该双特异性重组蛋白对非目标靶细胞的至少700倍。
表4不同接头序列的双特异性重组蛋白增殖抑制相对活性
Figure BDA0003268638110000441
实施例7靶向PD-L1双特异性重组蛋白增殖抑制活性检测
靶向PD-L1的双特异性重组蛋白对不同肿瘤细胞系的增殖抑制活性通过celltiter glo试剂盒(Promega，Cat:G7558)进行测定。以下方法以LCB-013、LCB-014、LCB-015为例，适用于第一功能结合片段结合PD-L1抗原、第二功能结合片段为IFN-α2b或其低亲和突变体的双特异性重组蛋白。
PD-L1阳性细胞系MDA-MB-231(购自南京科佰生物科技有限公司)铺板于96孔黑底透明板内(Corning，3904)，加入双特异性重组蛋白或对照蛋白(52nM起始，10倍梯度稀释，共6个浓度点)，放置二氧化碳培养箱37℃培养3天，加入Cell titer glo，Multomode PlateReader(PerkinElmer，Envision2105)检测信号值。
如图15所示，对于PD-L1阳性(PD-L1+)的目标靶细胞MDA-MB-231，靶向PD-L1的双特异性重组蛋白(LCB-013、LCB-014、LCB-015)都具有增殖抑制活性，并且活性显著高于不具有PD-L1靶向功能的对照双特异性重组蛋白LCB-012以及IFN-α2b，表明靶向PD-L1可以显著地增强IFN-α2b增殖抑制活性。表明拥有PD-L1靶向功能的双特异性重组蛋白其与目标靶细胞MDA-MB-231上PD-L1结合，可以显著地增强IFN-α2b的增殖抑制活性，而不具有目标靶细胞靶向作用的双特异性重组蛋白其IFN-α2b所起到的增殖抑制活性较低，揭示本发明双特异性重组蛋白仅对具有目的抗原的目标靶细胞有较强的增殖抑制作用，而对于不表达目的抗原的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋白的安全性较高。
PD-L1阳性的目标靶细胞MDA-MB-231铺板于96孔黑底透明板内(Corning，3904)，加入PD-L1抗体Atezolizumab或者同型对照200nM，37℃培养30分钟。加入双特异性重组蛋白或对照蛋白(起始浓度20nM，6倍稀释，共6个浓度点)，放置二氧化碳培养箱37℃培养3天，加入Cell titer glo，Multomode Plate Reader(PerkinElmer，Envision2105)检测信号值。
如图16所示，加入Atezolizumab封闭PD-L1抗原与抗体的结合位点后，LCB-015增殖抑制活性显著降低，该结果证明封闭待测细胞上目的抗原的结合功能，双特异性重组蛋白对待测细胞的增殖抑制活性显著下降，从另一个角度论证了本发明双特异性重组蛋白对于不具有目的抗原结合能力的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋白的安全性较高。
实施例8靶向CD38双特异性重组蛋白增殖抑制活性检测
靶向CD38的双特异性重组蛋白对不同肿瘤细胞系的增殖抑制活性通过celltiter glo试剂盒(Promega,Cat:G7558)进行测定。以下方法以LCB-016为例，适用于第一功能结合片段结合CD38抗原、第二功能结合片段为IFNα2b的双特异性重组蛋白的检测。
CD38阳性细胞系Daudi或者CD38阴性肿瘤细胞系SK-BR-3(购自南京科佰生物科技有限公司)铺板于96孔黑底透明板内(Corning,3904)，加入双特异性重组蛋白或对照蛋白(2nM起始，5倍梯度稀释，共9个点)，放置二氧化碳培养箱37℃培养3天，加入Cell titerglo，Multomode Plate Reader(PerkinElmer,Envision2105)检测信号值。
结果显示，双特异性重组蛋白(如LCB-016)对CD38阳性(CD38+)的目标靶细胞Daudi增殖抑制活性(IC50)非显而易见地强于对照IFN-α2b(如图17A所示)，相对的，双特异性重组蛋白(如LCB-016)对CD38阴性(CD38-)的非目标靶细胞SK-BR3增殖抑制活性(IC50)非显而易见地弱于对照IFN-α2b(如图17B所示)。即在增强第二功能结合片段为IFNα2b的双特异性重组蛋白中IFNα2b对具有目的抗原的目标靶细胞的增殖抑制活性的同时，显著降低双特异性重组蛋白中IFNα2b对不具有目的抗原的非目标靶细胞的增殖抑制作用。揭示本发明双特异性重组蛋白仅对具有目的抗原的目标靶细胞有较强的增殖抑制作用，而对于不具有目的抗原的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋白的安全性较高。
如图17A所示，对于CD38阳性(CD38+)的目标靶细胞Daudi，靶向CD38的双特异性重组蛋白(LCB-016)具有增殖抑制活性，并且活性显著高于不具有CD38靶向功能的IFN-α2b，表明靶向CD38可以显著地增强IFN-α2b增殖抑制活性。表明拥有CD38靶向功能的双特异性重组蛋白其与目标靶细胞Daudi上CD38结合，可以显著地增强IFN-α2b的增殖抑制活性。如图17B所示，缺乏CD38靶向功能的IFN-α2b对SK-BR3(CD38阴性的非目标靶细胞)的增殖抑制活性均显著强于拥有CD38靶向功能的双特异性重组蛋白LCB-016，表明双特异性重组蛋白其对不具有目的抗原的非目标靶细胞，其IFN-α2b所起到的增殖抑制活性较低。LCB-016在目标靶细胞上Daudi上对IFN-α2b的相对活性是其在非目标靶细胞SK-BR3上相对活性的至少200倍。
实施例9含IFN-α2b低亲和突变体双特异性重组蛋白增殖抑制活性检测
考虑到IFN-α2b的人体耐受剂量与常规抗体起效剂量间存在差异，为更好匹配目的抗原靶向的抗原结合片段与IFN-α2b的效果，实现高效低毒的效果，本发明还设计了一系列包含IFN-α2b低亲和突变体的双特异性重组蛋白。本实施例以基于LCB-010的突变设计为例设计含IFN-α2b低亲和突变体双特异性重组蛋白，检测含不同IFN-α2b低亲和突变体的双特异性重组蛋白的增殖抑制活性，实验方法同实施例7。
如图18-20所示，结果表明，拥有IFN-α2b低亲和突变体的双特异性重组蛋白在GPC3阳性的目标靶细胞(HuH-7)或GPC3阴性的非目标靶细胞(U266、SW480)，LCB-010-M2、LCB-010-M3和LCB-010-M4增殖抑制活性相对于LCB-010均有降低。例如，LCB-010-M3(A145G突变)在HuH-7(GPC3阳性细胞系，目标靶细胞)的增殖抑制活性IC50与IFN-α2b相当(IC50IFN-α2b＝0.1793，IC50LCB-010-M3＝0.179)，但LCB-010-M3在GPC3阴性的非目标靶细胞(U266)的增殖抑制相对活性(IC50 IFN-α2b/双特异性重组蛋白)更弱，例如，LCB-010-M3在U266上的增殖抑制相对活性(IC50 IFN-α2b/双特异性重组蛋白)为0.000615，显著弱于IFN-α2b，弱于LCB-010，LCB-010-M4(R149A突变)也有类似的结果。
以上结果及图19-图21表明含IFN-α2b低亲和突变体的靶向GPC3双特异性重组蛋白在目的抗原阳性的目标靶细胞系中活性与IFN-α2b相当，但在不表达目的抗原的非目标靶细胞系中活性降低，提示本发明含IFN-α2b低亲和突变体的双特异性重组蛋白更高的安全性。
综上，含IFN-α2b低亲和突变体的本发明双特异性重组蛋白，其对目的抗原阴性的非目标靶细胞的增殖抑制活性相较含IFN-α2b野生型双特异性重组蛋白对目的抗原阴性的非目标靶细胞的增殖抑制活性的下降程度，相较含IFN-α2b低亲和突变体双特异性重组蛋白在目的抗原阳性的目标靶细胞的下降程度相当或更显著。
实施例10双特异性重组蛋白潜在风险杂质对非目标靶细胞的增殖抑制活性检测
为降低双特异性重组蛋白未来制备工艺过程中潜在的杂质安全性风险，本发明以LCB-010为例，分析其潜在风险杂质(B链同二聚体)对非目标靶细胞的增殖抑制情况。
在LCB-010精纯过程(如实施例2所述)中，分离LCB-010的B链同二聚体(图3，同二聚体分子量约140kD)，检测该潜在风险杂质与IFN-α2b-Fc(CN108864290A中图3A所示双抗结构的潜在风险杂质，D1换成IFN-α2b后的右臂同二聚体)在非目标靶细胞(GPC3阴性细胞)MDA-MB-231中的增殖抑制活性。结果如图21显示，在16.7nM浓度下，IFN-α2b对MDA-MB-231(GPC3阴性细胞，非目标靶细胞)增殖抑制率为91.3％，IFN-α2b-Fc对MDA-MB-231(GPC3阴性细胞，非目标靶细胞)增殖抑制率为66.8％(较IFN-α2b下降约24.5％)，LCB-010的B链同二聚体对MDA-MB-231(GPC3阴性细胞，非目标靶细胞)增殖抑制率仅为16.2％(较IFN-α2b降低约75.1％，较IFN-α2b-Fc降低约50％)。综上，潜在风险杂质对非目标靶细胞仅有极弱的作用，即潜在的安全性风险或带来的潜在毒副作用极低。
实施例11第二功能结合片段为IL12的双特异性重组蛋白靶标亲和力检测
双特异性重组蛋白对靶标TIGIT和IL12受体的结合亲和力通过流式细胞术进行测定。以下方法以LCB-018为例，适用于第一功能抗原为TIGIT，第二功能结合片段为IL12A的重组蛋白的检测。
H_IL12 Reporter 293细胞(TIGIT+，目标靶细胞)铺于96孔白壁底透明板内(Corning，3903)，贴壁过夜后，弃掉培养基，加入抗TIGIT单抗Tiragolumab或者同种型对照抗体(Isotype)200nM，37℃培养30分钟后弃掉培养基，将200nM双特异性重组蛋白LCB-018和2μg/mL的IL12B等体积混匀后，3倍向下稀释，共10个浓度点，然后150μL/well加入96孔板中，将其放置二氧化碳培养箱37℃培养6h，加入One-Glo，Multomode Plate Reader(PerkinElmer，Envision2105)检测信号值。
结果显示(如图22所示)，加入抗TIGIT单抗Tiragolumab封闭，双特异性重组蛋白与待测细胞的结合曲线明显右移，证明本发明双特异性重组蛋白通过合适接头序列连接第一功能结构片段(TIGIT靶向部分)和第二功能结构片段(IL12)的重组蛋白可非显而易见地提高双特异性重组蛋白与目标靶细胞的结合能力。此外，针对目的抗原阳性的目标靶细胞，本发明双特异性重组蛋白(如LCB-018)对该目标靶细胞(如H_IL12 Reporter 293细胞)的结合亲和力比同浓度下的IL12A与IL12B复合物对同细胞的结合亲和力弱约500倍左右(EC50LCB-018＝30nM，EC50IL12A/IL12B＝0.06nM)，也证明了本发明双特异性重组蛋白其靶向作用可显著降低IL12与细胞的结合活性，降低IL12潜在的毒副作用，尤其是IL12与非目标靶细胞结合产生的毒副作用。
实施例12第二功能结合片段为IL10M的双特异性重组蛋白对THP1细胞P-STAT3激活水平检测
双特异性重组蛋白对THP1细胞P-STAT3激活水平检测通过流式细胞术进行测定。以下方法以LCB-019为例，适用于第一功能抗原为CD80，第二功能结合片段为IL10M的重组蛋白的检测。
用基础培养基(1640)将LCB-019、LCB-022(无CD80靶向作用的对照双特异性重组蛋白)、Isotype、IL10M-Fc融合蛋白以等摩尔浓度106nM进行稀释备用。
考虑到LPS(sigma,L5418-2ML)可以刺激Thp1细胞(来源中科院细胞库)表面CD80抗原的表达，本实施例以LPS刺激24h后的Thp1细胞模拟目的CD80阳性的双阳细胞(目标靶细胞)。
将1μg/mL的LPS(sigma,L5418-2ML)刺激24h后的Thp1细胞(来源中科院细胞库)以1×106密度重悬于基础培养基(1640)中。将LPS刺激后的重悬细胞、未被LPS刺激的Thp1细胞以100ul体积铺于96孔板中，并分别加入等体积稀释好的LCB-019、LCB-022(对照双特异性重组蛋白)、Isotype、IL10M-Fc融合蛋白在37℃、5％二氧化碳培养箱中孵育20分钟。孵育完毕后，离心去上清，并对细胞进行固定和破膜处理。然后将细胞重悬于100μL含0.5μLPE-P-STAT3抗体(BD,562072)的FACS buffer(1×PBS+2％FBS)中避光4℃孵育1h。FACS buffer(1×PBS+2％FBS)清洗两次后，加入200μL FACS buffer(1×PBS+2％FBS)重悬，用FACS检测P-STAT3水平。
结果显示，如图23所示，在经过LPS刺激后高表达目的抗原CD80的Thp1细胞上，具有目的抗原(CD80)靶向作用的双特异性重组蛋白LCB-019的P-STAT3水平基本与IL10M-Fc融合蛋白相当，显著高于未有目的抗原靶向功能的对照双特异性重组蛋白LCB-022，而在未经LPS刺激的Thp1细胞上，LCB-019和对照双特异性重组蛋白LCB-022其P-STAT3水平基本相当，且明显弱于IL10M-Fc融合蛋白。
可见，针对目的抗原表达较弱或不表达目的抗原的非目标靶细胞，双特异性重组蛋白对非目标靶细胞的作用显著弱于IL10M-Fc融合蛋白，展现出其相对更高的安全性，而针对目的抗原高表达的目标靶细胞，具有目的抗原靶向的双特异性重组蛋白可展现出与IL10M-Fc融合蛋白相当的STAT3水平，充分发挥并提升双特异性重组蛋白中IL10M的功效与作用，相对的，不具有目的抗原靶向功能的双特异性重组蛋白则与该细胞(非目标靶细胞)的作用相对较弱，表现出较佳的安全性。
实施例13第二功能结合片段为IL15-IL15RαSUSHI的双特异性重组蛋白增殖活性检测
双特异性重组蛋白对OKT3刺激48小时后PD-1阳性的hPBMC的增殖活性检测通过流式细胞术进行测定。以下方法以LCB-020、LCB-021、LCB-023、LCB-024为例，适用于第一功能抗原为PD-1，第二功能结合片段为IL15-IL15RαSUSHI的重组蛋白的检测。
将hPBMC细胞复苏后放入预包被好100ng/mL anti-CD3抗体(OKT3,eBioscience,Cat.#16-0037-85)的6孔板中，孵育48小时。离心收集活化的PBMC并用PBS洗一遍。随后用培养基重悬细胞，按照1.5E5/100μL/孔的密度铺到96孔板中，并加入1.6nM的阳性对照C15Y、双特异性重组蛋白LCB-020、LCB-021、LCB-023和LCB-024。96孔板放置于37℃的二氧化碳培养箱中孵育96小时。孵育结束后，为了细胞分类，首先用anti-CD4-APC(eBioscience,Cat.#17-0049-42)和anti-CD8-FITC(invitrogen,Cat.#MHCD0801)抗体对细胞膜进行第一次染色。染色结束后，用固定透膜试剂处理细胞使细胞透膜化(eBioscienceTMFoxp3/Transcription Factor Staining Buffer Set.Invitrogen,Cat.#00-5523-00)。透膜结束后，用anti-Ki-67-PE(Biolegend,Cat.#350504)抗体对细胞染色40分钟。染色结束用FACS分析CD4+和CD8+细胞群上Ki-67的表达。
如图24所示，5个样品在1.6nM浓度下对PD-1高表达的hPBMC均有一定的促增殖效果，其中，阳性对照C15Y活性最强，具有PD-1靶向作用的双特异性重组蛋白LCB-020、LCB-021和不具有PD-1靶向作用的对照双特异性重组蛋白RSV×IL-15R抗体(LCB-023、LCB-024)促增殖效果弱于阳性对照C15Y，且具有PD-1靶向作用的双特异性重组蛋白LCB-020、LCB-021其促增殖效果明显强于不具有PD-1靶向作用的对照双特异性重组蛋白LCB-023、LCB-024，同时，不具有PD-1靶向作用的对照双特异性重组蛋白LCB-023、LCB-024其促增殖效果较弱。结果显示具有目的抗原靶向功能的双特异性重组蛋白可显著提升IL15-IL15RαSUSHI对目标靶细胞的促增殖效果，且该促增殖效果弱于IL15-IL15RαSUSHI-Fc融合蛋白(C15Y)；对照双特异性重组蛋白对(LCB-023、LCB-024)对OKT3刺激24h后PD-1阳性的hPBMC的弱促增殖效果，则从另一个侧面揭示本发明双特异性重组蛋白对于不表达目的抗原的非目标靶细胞作用较弱，证明其安全性较高。
靶向PD-1的双特异性重组蛋白对M-07e的增殖活性通过cell titer glo试剂盒(Promega,Cat:G7558)进行测定。以下方法以LCB-020、LCB-021、LCB-023、LCB-024为例，适用于第一功能抗原为PD-1，第二功能结合片段为IL15-IL15RαSUSHI的重组蛋白的检测。
收集细胞因子依赖性细胞M-07e并用PBS洗一遍。用不含GM-CSF(R&D,Cat.#215-GM-050)生长因子的培养基将细胞稀释成2E4/50μL的密度铺到96孔板中，孵育4小时用于细胞因子饥饿。4小时饥饿处理后，加入梯度稀释的C15Y(10nM起始浓度，3倍稀释)、双特异性重组蛋白LCB-020、LCB-021、LCB-023和LCB-024(333nM起始浓度，3倍稀释)，在37℃的二氧化碳培养箱中共孵育72小时。72小时后，用Celltiter-glo(Promega,Cat.#G7573)检测活细胞数量。
如图25所示，阳性对照C15Y在极低的浓度下即发挥出极强的促增殖效果(EC50＝0.05267nM)，而本发明双特异性重组蛋白则需在相对较高的浓度下才发挥出促增殖效果(EC50约在21-37nM之间)，且EC50无显著差异，且由于待测细胞M-07e不表达LCB-020、LCB-021、LCB-023和LCB-024对应的目的抗原，因此M-07e可视为非目标靶细胞，第二功能结合片段为IL15-IL15RαSUSHI的本发明双特异性重组蛋白对非目标靶细胞的促增殖效果需在较高的浓度下才能发挥功效，EC50相较阳性对照高出400倍以上。
综上可见，本发明双特异性重组蛋白对于目的抗原阳性的目标靶细胞，具有靶向功能的双特异性重组蛋白其促增殖效果明显强于不具有靶向功能的双特异性重组蛋白，但弱于阳性对照C15Y；相反的，本发明重组蛋白对于目的抗原阴性的非目标靶细胞，其促增殖效果未见显著差异，且促增殖效果的起效浓度显著高于阳性对照C15Y。可见，本发明具有靶向功能的双特异性重组蛋白对于目标靶细胞在相对较低的浓度下即可发挥促增殖效果，但对于非目标靶细胞则需在极高的浓度下才能发挥促增殖效果。
实施例14双特异性重组蛋白冻融稳定性考察
现有的重组人白蛋白干扰素-α2b融合蛋白冻融稳定性较差，不宜反复冻融，如PEG化的长效干扰素不可冷冻和震荡，对运输和保存条件要求较高。为了研究本发明双特异性重组蛋白的冻融稳定性，对双特异性重组蛋白进行反复冻融稳定性测试。蛋白放置于20mMNaAc(PH＝5)缓冲液，在-40℃条件下进行5次的反复冻融。对冻融前后的样品进行纯度(尺寸排阻层析，SEC)和外观分析。结果如表5所示，LCB-011及其突变体冻融稳定性良好，纯度均在95％以上，5次反复冻融后外观澄清，表明本发明双特异性重组蛋白其IFN-α2b蛋白部分冻融稳定性明显优于IFN-α2b单体或者PEG化的IFN-α2b。
表5双特异性重组蛋白冻融稳定性
Figure BDA0003268638110000511
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。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
SEQUENCE LISTING
<110> 上海霖羲致企业管理有限公司
<120> 双特异性重组蛋白及其用途
<130> P21016874CN
<150> CN2020109777162
<151> 2020-09-17
<150> CN2021109331052
<151> 2021-08-12
<160> 64
<170> PatentIn version 3.5
<210> 1
<211> 452
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofatumumab(H)-Fc1
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 2
<211> 558
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofatumumab(L)-SIRPαD1-Fc2
<400> 2
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Glu Glu Glu Leu Gln Val Ile Gln Pro Asp
210 215 220
Lys Ser Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr
225 230 235 240
Val Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala
245 250 255
Gly Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro
260 265 270
Arg Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe
275 280 285
Ser Ile Ser Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr
290 295 300
Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly
305 310 315 320
Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Asp Lys Thr His Thr
325 330 335
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
340 345 350
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
355 360 365
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
370 375 380
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
385 390 395 400
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
405 410 415
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
420 425 430
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
435 440 445
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
450 455 460
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
465 470 475 480
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
485 490 495
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
500 505 510
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
515 520 525
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
530 535 540
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550 555
<210> 3
<211> 573
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofatumumab(L)-(GGGGS)3-SIRPαD1-Fc2
<400> 3
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys
225 230 235 240
Ser Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val
245 250 255
Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly
260 265 270
Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg
275 280 285
Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser
290 295 300
Ile Ser Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys
305 310 315 320
Val Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala
325 330 335
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Asp Lys Thr His Thr Cys
340 345 350
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
355 360 365
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
370 375 380
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
385 390 395 400
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
405 410 415
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
420 425 430
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
435 440 445
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
450 455 460
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
465 470 475 480
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
485 490 495
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
500 505 510
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
515 520 525
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
530 535 540
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
545 550 555 560
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 4
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(H)-Fc1
<400> 4
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 5
<211> 616
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)1-IFNα 2b-Fc2
<400> 5
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
225 230 235 240
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
245 250 255
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
260 265 270
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
275 280 285
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
290 295 300
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
305 310 315 320
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
325 330 335
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
340 345 350
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
355 360 365
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
370 375 380
Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
385 390 395 400
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
405 410 415
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
420 425 430
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
435 440 445
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
450 455 460
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
465 470 475 480
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
485 490 495
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
500 505 510
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
515 520 525
Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
530 535 540
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
545 550 555 560
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
565 570 575
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
580 585 590
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
595 600 605
Ser Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 6
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)2-IFNα 2b-Fc2
<400> 6
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
245 250 255
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 7
<211> 631
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)4-IFNα 2b-Fc2
<400> 7
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys
225 230 235 240
Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
245 250 255
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg
260 265 270
His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
275 280 285
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
290 295 300
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu
305 310 315 320
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
325 330 335
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu
340 345 350
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
355 360 365
Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala
370 375 380
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu
385 390 395 400
Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
405 410 415
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
420 425 430
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
435 440 445
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
450 455 460
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
465 470 475 480
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
485 490 495
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
500 505 510
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
515 520 525
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
530 535 540
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
545 550 555 560
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
565 570 575
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
580 585 590
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
595 600 605
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
610 615 620
Leu Ser Leu Ser Pro Gly Lys
625 630
<210> 8
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)2-IFNα 2b（L26A）-Fc2
<400> 8
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Ala Phe
245 250 255
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 9
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)2-IFNα 2b（L30A）-Fc2
<400> 9
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
245 250 255
Ser Cys Ala Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 10
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)2-IFNα 2b（A145G）-Fc2
<400> 10
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
245 250 255
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Gly Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 11
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)2-IFNα 2b（R149A）-Fc2
<400> 11
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
245 250 255
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Ala Ser Phe Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 12
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)2-IFNα 2b（S152A）-Fc2
<400> 12
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
245 250 255
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ala Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 13
<211> 631
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)4-IFNα 2b（A145G）-Fc2
<400> 13
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys
225 230 235 240
Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
245 250 255
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg
260 265 270
His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
275 280 285
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
290 295 300
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu
305 310 315 320
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
325 330 335
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu
340 345 350
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
355 360 365
Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Gly
370 375 380
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu
385 390 395 400
Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
405 410 415
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
420 425 430
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
435 440 445
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
450 455 460
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
465 470 475 480
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
485 490 495
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
500 505 510
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
515 520 525
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
530 535 540
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
545 550 555 560
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
565 570 575
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
580 585 590
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
595 600 605
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
610 615 620
Leu Ser Leu Ser Pro Gly Lys
625 630
<210> 14
<211> 631
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)-(GGGGS)4-IFNα 2b（R149A）-Fc2
<400> 14
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys
225 230 235 240
Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
245 250 255
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg
260 265 270
His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
275 280 285
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
290 295 300
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu
305 310 315 320
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
325 330 335
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu
340 345 350
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
355 360 365
Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala
370 375 380
Glu Ile Met Ala Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu
385 390 395 400
Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
405 410 415
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
420 425 430
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
435 440 445
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
450 455 460
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
465 470 475 480
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
485 490 495
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
500 505 510
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
515 520 525
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
530 535 540
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
545 550 555 560
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
565 570 575
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
580 585 590
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
595 600 605
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
610 615 620
Leu Ser Leu Ser Pro Gly Lys
625 630
<210> 15
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Palivizumab(H)- Fc1
<400> 15
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45
Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80
Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 16
<211> 615
<212> PRT
<213> Artificial Sequence
<220>
<223> Palivizumab (L)-(GGGGS)2-IFNα 2b-Fc2
<400> 16
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys
210 215 220
Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
225 230 235 240
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg
245 250 255
His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
260 265 270
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
275 280 285
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu
290 295 300
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
305 310 315 320
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu
325 330 335
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
340 345 350
Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala
355 360 365
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu
370 375 380
Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
385 390 395 400
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
405 410 415
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
420 425 430
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
435 440 445
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
450 455 460
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
465 470 475 480
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
485 490 495
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
500 505 510
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
515 520 525
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
530 535 540
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
545 550 555 560
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
565 570 575
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
580 585 590
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
595 600 605
Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 17
<211> 415
<212> PRT
<213> Artificial Sequence
<220>
<223> IFN-α 2b-Fc1
<400> 17
Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys
1 5 10 15
Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu
20 25 30
Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser
35 40 45
Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu
50 55 60
Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His
65 70 75 80
Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser
85 90 95
Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
100 105 110
Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val
115 120 125
Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys
130 135 140
Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro
145 150 155 160
Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
165 170 175
Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His
180 185 190
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
195 200 205
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
210 215 220
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
225 230 235 240
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
245 250 255
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
260 265 270
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
275 280 285
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
290 295 300
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
305 310 315 320
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
325 330 335
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
340 345 350
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
355 360 365
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
370 375 380
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
385 390 395 400
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
405 410 415
<210> 18
<211> 188
<212> PRT
<213> Artificial Sequence
<220>
<223> IFN-α 2b
<400> 18
Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys
1 5 10 15
Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu
20 25 30
Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser
35 40 45
Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu
50 55 60
Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His
65 70 75 80
Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser
85 90 95
Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
100 105 110
Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val
115 120 125
Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys
130 135 140
Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro
145 150 155 160
Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
165 170 175
Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
180 185
<210> 19
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(H)-Fc
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 20
<211> 219
<212> PRT
<213> Artificial Sequence
<220>
<223> GC33(L)
<400> 20
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 21
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab（H）-Fc1
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 22
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab（L）
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 23
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab (H)-Fc1
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
355 360 365
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 24
<211> 611
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab (L)-(GGGGS)1-IFNα 2b-Fc2
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln
210 215 220
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met
225 230 235 240
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
245 250 255
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile
260 265 270
Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr
275 280 285
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr
290 295 300
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln
305 310 315 320
Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu
325 330 335
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys
340 345 350
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg
355 360 365
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
370 375 380
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
385 390 395 400
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
405 410 415
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
420 425 430
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
435 440 445
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
450 455 460
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
465 470 475 480
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
485 490 495
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
500 505 510
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
515 520 525
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
530 535 540
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
545 550 555 560
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
565 570 575
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
580 585 590
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
595 600 605
Pro Gly Lys
610
<210> 25
<211> 616
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab (L)-(GGGGS)2-IFNα 2b-Fc2
<400> 25
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
225 230 235 240
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
245 250 255
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
260 265 270
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
275 280 285
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
290 295 300
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
305 310 315 320
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
325 330 335
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
340 345 350
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
355 360 365
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
370 375 380
Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
385 390 395 400
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
405 410 415
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
420 425 430
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
435 440 445
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
450 455 460
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
465 470 475 480
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
485 490 495
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
500 505 510
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
515 520 525
Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
530 535 540
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
545 550 555 560
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
565 570 575
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
580 585 590
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
595 600 605
Ser Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 26
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> Atezolizumab (L)-(GGGGS)3-IFNα 2b-Fc2
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
225 230 235 240
Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
245 250 255
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
260 265 270
Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
275 280 285
Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
290 295 300
Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
305 310 315 320
Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu
325 330 335
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
340 345 350
Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
355 360 365
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr
370 375 380
Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 27
<211> 446
<212> PRT
<213> Artificial Sequence
<220>
<223> Edrecolomab (H)-Fc1
<400> 27
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr
20 25 30
Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Asp Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 28
<211> 583
<212> PRT
<213> Artificial Sequence
<220>
<223> Edrecolomab (L)-(GGGGS)5- SIRPαD1-Fc2
<400> 28
Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly
1 5 10 15
Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
225 230 235 240
Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
245 250 255
Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val
260 265 270
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
275 280 285
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
290 295 300
Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala Ile
305 310 315 320
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
325 330 335
Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val
340 345 350
Arg Ala Lys Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
355 360 365
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
370 375 380
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
385 390 395 400
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
405 410 415
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
420 425 430
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
435 440 445
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
450 455 460
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
465 470 475 480
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
485 490 495
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
500 505 510
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
515 520 525
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
530 535 540
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
545 550 555 560
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
565 570 575
Leu Ser Leu Ser Pro Gly Lys
580
<210> 29
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> SWA11(H)-Fc1
<400> 29
Asp Val His Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Thr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Thr Val Glu Trp
35 40 45
Met Gly Tyr Ile Gln Tyr Thr Gly Ser Thr Arg Tyr Asn Pro Ala Leu
50 55 60
Arg Gly Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Ile Ser Val Thr Thr Ala Asp Thr Gly Thr Tyr Phe Cys
85 90 95
Ala Arg Gly Thr Thr Ala Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Leu Thr Val Ala Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
355 360 365
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 30
<211> 574
<212> PRT
<213> Artificial Sequence
<220>
<223> SWA11(L)-(GGGGS)2- SIRPαD1-Fc2
<400> 30
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Asn Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Arg Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Ala Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Gly Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Phe Ile Tyr Pro Leu Thr Phe Gly Val Gly Thr Lys Leu Gly Leu
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp
225 230 235 240
Lys Ser Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr
245 250 255
Val Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala
260 265 270
Gly Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro
275 280 285
Arg Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe
290 295 300
Ser Ile Ser Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr
305 310 315 320
Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly
325 330 335
Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
465 470 475 480
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 31
<211> 444
<212> PRT
<213> Artificial Sequence
<220>
<223> Mezagitamab(L)-Fc1
<400> 31
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn
20 25 30
Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asp Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu
85 90 95
Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser Cys Asp Lys Thr His Thr Cys Pro
210 215 220
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
340 345 350
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 32
<211> 643
<212> PRT
<213> Artificial Sequence
<220>
<223> Mezagitamab (H)-(GGGGS)5-IFNα2b-Fc2
<400> 32
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Asp Ile Ser Trp Asn Gly Gly Lys Thr His Tyr Val Asp Ser Val
50 55 60
Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220
Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln
245 250 255
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met
260 265 270
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly
275 280 285
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile
290 295 300
Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr
305 310 315 320
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr
325 330 335
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln
340 345 350
Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu
355 360 365
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys
370 375 380
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg
385 390 395 400
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
405 410 415
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
420 425 430
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
435 440 445
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
450 455 460
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
465 470 475 480
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
485 490 495
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
500 505 510
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
515 520 525
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
530 535 540
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
545 550 555 560
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
565 570 575
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
580 585 590
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
595 600 605
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
610 615 620
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
625 630 635 640
Pro Gly Lys
<210> 33
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Felzartamab(H)-Fc2
<400> 33
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asp Pro Ser Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 34
<211> 582
<212> PRT
<213> Artificial Sequence
<220>
<223> Felzartamab(L)-(GGGGS)5- SIRPαD1-Fc1
<400> 34
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg His Tyr Tyr Val
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Thr Gly Gly Ala Ser Leu
85 90 95
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala
100 105 110
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala
115 120 125
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala
130 135 140
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val
145 150 155 160
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser
165 170 175
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr
180 185 190
Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala
195 200 205
Pro Thr Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Glu
225 230 235 240
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly
245 250 255
Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly
260 265 270
Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr
275 280 285
Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser
290 295 300
Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala Ile Thr
305 310 315 320
Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
325 330 335
Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg
340 345 350
Ala Lys Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
355 360 365
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
370 375 380
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
385 390 395 400
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
405 410 415
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
420 425 430
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
435 440 445
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
450 455 460
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
465 470 475 480
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
485 490 495
Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
500 505 510
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
515 520 525
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
530 535 540
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
545 550 555 560
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
565 570 575
Ser Leu Ser Pro Gly Lys
580
<210> 35
<211> 456
<212> PRT
<213> Artificial Sequence
<220>
<223> Tiragolumab(H)-Fc1
<400> 35
Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Lys Thr Tyr Tyr Arg Phe Lys Trp Tyr Ser Asp Tyr Ala
50 55 60
Val Ser Val Lys Gly Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Phe Tyr Cys Thr Arg Glu Ser Thr Thr Tyr Asp Leu Leu Ala Gly Pro
100 105 110
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
115 120 125
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
165 170 175
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
180 185 190
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
225 230 235 240
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
290 295 300
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
305 310 315 320
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
340 345 350
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
355 360 365
Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
385 390 395 400
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
405 410 415
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 36
<211> 664
<212> PRT
<213> Artificial Sequence
<220>
<223> Tiragolumab (L)-(GGGGS)4-IL12A-Fc2
<400> 36
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
225 230 235 240
Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu
245 250 255
His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys
260 265 270
Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp
275 280 285
His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu
290 295 300
Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr
305 310 315 320
Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe
325 330 335
Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr
340 345 350
Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys
355 360 365
Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu
370 375 380
Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser
385 390 395 400
Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu
405 410 415
Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser
420 425 430
Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
435 440 445
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
450 455 460
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
465 470 475 480
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
485 490 495
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
500 505 510
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
515 520 525
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
530 535 540
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
545 550 555 560
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
565 570 575
Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
580 585 590
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
595 600 605
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
610 615 620
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
625 630 635 640
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
645 650 655
Ser Leu Ser Leu Ser Pro Gly Lys
660
<210> 37
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> Galiximab(H)-Fc1
<400> 37
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Gly Gly
20 25 30
Tyr Gly Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
35 40 45
Ile Gly Ser Phe Tyr Ser Ser Ser Gly Asn Thr Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Gln Val Thr Ile Ser Thr Asp Thr Ser Lys Asn Gln Phe
65 70 75 80
Ser Leu Lys Leu Asn Ser Met Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Val Arg Asp Arg Leu Phe Ser Val Val Gly Met Val Tyr Asn Asn
100 105 110
Trp Phe Asp Val Trp Gly Pro Gly Val Leu Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
355 360 365
Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 38
<211> 634
<212> PRT
<213> Artificial Sequence
<220>
<223> Galiximab(L)- (GGGGS)5-IL10M-Fc2
<400> 38
Glu Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Lys Val Thr Ile Ser Cys Thr Gly Ser Thr Ser Asn Ile Gly Gly Tyr
20 25 30
Asp Leu His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Ile Asn Lys Arg Pro Ser Gly Ile Ser Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ala Ala Ser Leu Ala Ile Thr Gly Leu Gln
65 70 75 80
Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu
85 90 95
Asn Ala Gln Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe
245 250 255
Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser
260 265 270
Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu
275 280 285
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln
290 295 300
Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln
305 310 315 320
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly
325 330 335
Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
340 345 350
Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val
355 360 365
Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr
370 375 380
Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
385 390 395 400
Met Thr Met Lys Ile Arg Asn Asp Lys Thr His Thr Cys Pro Pro Cys
405 410 415
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
420 425 430
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
435 440 445
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
450 455 460
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
465 470 475 480
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
485 490 495
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
500 505 510
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
515 520 525
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
530 535 540
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
545 550 555 560
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
565 570 575
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
580 585 590
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
595 600 605
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
610 615 620
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
625 630
<210> 39
<211> 443
<212> PRT
<213> Artificial Sequence
<220>
<223> Nivolumab(H)-Fc1
<400> 39
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
210 215 220
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
290 295 300
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
305 310 315 320
Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
325 330 335
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
340 345 350
Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe
355 360 365
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
370 375 380
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
385 390 395 400
Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
405 410 415
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 40
<211> 675
<212> PRT
<213> Artificial Sequence
<220>
<223> Nivolumab(L)-(GGGGS)5-IL15-(GGGGS)5- IL15RαSUSHI-Fc2
<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn
225 230 235 240
Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln
245 250 255
Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro
260 265 270
Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val
275 280 285
Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn
290 295 300
Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr
305 310 315 320
Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys
325 330 335
Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr
340 345 350
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
355 360 365
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro
370 375 380
Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr
385 390 395 400
Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly
405 410 415
Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala
420 425 430
His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Glu Pro Lys Ser Ser
435 440 445
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
450 455 460
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
465 470 475 480
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
485 490 495
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
500 505 510
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
515 520 525
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
530 535 540
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
545 550 555 560
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
565 570 575
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
580 585 590
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
595 600 605
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
610 615 620
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
625 630 635 640
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
645 650 655
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
660 665 670
Pro Gly Lys
675
<210> 41
<211> 675
<212> PRT
<213> Artificial Sequence
<220>
<223> Nivolumab(L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-Fc2
<400> 41
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile
225 230 235 240
Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys
245 250 255
Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe
260 265 270
Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys
275 280 285
Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg
290 295 300
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
305 310 315 320
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser
325 330 335
Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala
340 345 350
Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala
355 360 365
Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly
370 375 380
Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn
385 390 395 400
Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu
405 410 415
Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe
420 425 430
Val His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser Ser
435 440 445
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
450 455 460
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
465 470 475 480
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
485 490 495
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
500 505 510
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
515 520 525
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
530 535 540
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
545 550 555 560
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
565 570 575
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
580 585 590
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
595 600 605
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
610 615 620
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
625 630 635 640
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
645 650 655
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
660 665 670
Pro Gly Lys
675
<210> 42
<211> 631
<212> PRT
<213> Artificial Sequence
<220>
<223> Palivizumab (L)- (GGGGS)5-IL10M-Fc2
<400> 42
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro
225 230 235 240
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn
245 250 255
Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys
260 265 270
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu
275 280 285
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser
290 295 300
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
305 310 315 320
Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
325 330 335
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys
340 345 350
Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Val
355 360 365
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met
370 375 380
Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met
385 390 395 400
Lys Ile Arg Asn Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
405 410 415
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
420 425 430
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
435 440 445
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
450 455 460
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
465 470 475 480
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
485 490 495
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
500 505 510
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
515 520 525
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
530 535 540
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
545 550 555 560
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
565 570 575
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
580 585 590
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
595 600 605
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
610 615 620
Leu Ser Leu Ser Pro Gly Lys
625 630
<210> 43
<211> 674
<212> PRT
<213> Artificial Sequence
<220>
<223> Palivizumab(L)-(GGGGS)5-IL15-(GGGGS)5- IL15RαSUSHI-Fc2
<400> 43
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp
225 230 235 240
Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser
245 250 255
Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser
260 265 270
Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile
275 280 285
Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu
290 295 300
Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu
305 310 315 320
Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu
325 330 335
Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser
340 345 350
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
355 360 365
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met
370 375 380
Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser
385 390 395 400
Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr
405 410 415
Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His
420 425 430
Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Glu Pro Lys Ser Ser Asp
435 440 445
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
450 455 460
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
465 470 475 480
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
485 490 495
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
500 505 510
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
515 520 525
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
530 535 540
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
545 550 555 560
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
565 570 575
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
580 585 590
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
595 600 605
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
610 615 620
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
625 630 635 640
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
645 650 655
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
660 665 670
Gly Lys
<210> 44
<211> 674
<212> PRT
<213> Artificial Sequence
<220>
<223> Palivizumab (L)-(GGGGS)5-IL15RαSUSHI-(GGGGS)5-IL15-Fc2
<400> 44
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr
225 230 235 240
Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser
245 250 255
Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys
260 265 270
Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala
275 280 285
Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Gly
290 295 300
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
305 310 315 320
Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile Ser Asp
325 330 335
Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr
340 345 350
Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met
355 360 365
Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp
370 375 380
Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn
385 390 395 400
Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys
405 410 415
Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val
420 425 430
His Ile Val Gln Met Phe Ile Asn Thr Ser Glu Pro Lys Ser Ser Asp
435 440 445
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
450 455 460
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
465 470 475 480
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
485 490 495
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
500 505 510
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
515 520 525
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
530 535 540
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
545 550 555 560
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
565 570 575
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
580 585 590
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
595 600 605
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
610 615 620
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
625 630 635 640
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
645 650 655
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
660 665 670
Gly Lys
<210> 45
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Felzartamab(H)-Fc
<400> 45
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asp Pro Ser Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Leu Pro Leu Val Tyr Thr Gly Phe Ala Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 46
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> Felzartamab(L)
<400> 46
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg His Tyr Tyr Val
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Thr Gly Gly Ala Ser Leu
85 90 95
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala
100 105 110
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala
115 120 125
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala
130 135 140
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val
145 150 155 160
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser
165 170 175
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr
180 185 190
Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala
195 200 205
Pro Thr Glu Cys Ser
210
<210> 47
<211> 393
<212> PRT
<213> Artificial Sequence
<220>
<223> IL10M-Fc
<400> 47
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met
145 150 155 160
Thr Met Lys Ile Arg Asn Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Lys
385 390
<210> 48
<211> 322
<212> PRT
<213> Artificial Sequence
<220>
<223> IL12B
<400> 48
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
20 25 30
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
35 40 45
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
50 55 60
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly
65 70 75 80
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
85 90 95
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
100 105 110
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
115 120 125
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
130 135 140
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
145 150 155 160
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
165 170 175
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
180 185 190
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
195 200 205
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
210 215 220
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
225 230 235 240
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
245 250 255
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
260 265 270
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
275 280 285
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
290 295 300
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
305 310 315 320
Cys Ser
<210> 49
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> 信号肽
<400> 49
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala
<210> 50
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> SIRPαD1
<400> 50
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> 51
<211> 197
<212> PRT
<213> Artificial Sequence
<220>
<223> IL-12A
<400> 51
Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu
1 5 10 15
His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys
20 25 30
Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp
35 40 45
His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu
50 55 60
Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr
65 70 75 80
Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe
85 90 95
Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr
100 105 110
Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys
115 120 125
Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu
130 135 140
Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser
145 150 155 160
Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu
165 170 175
Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser
180 185 190
Tyr Leu Asn Ala Ser
195
<210> 52
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> IL-10M
<400> 52
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu
115 120 125
Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
130 135 140
Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met
145 150 155 160
Thr Met Lys Ile Arg Asn
165
<210> 53
<211> 114
<212> PRT
<213> Artificial Sequence
<220>
<223> IL-15
<400> 53
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
1 5 10 15
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
20 25 30
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
35 40 45
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
50 55 60
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
65 70 75 80
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
85 90 95
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
100 105 110
Thr Ser
<210> 54
<211> 65
<212> PRT
<213> Artificial Sequence
<220>
<223> IL-15RαSUSHI
<400> 54
Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val
1 5 10 15
Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly
20 25 30
Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn
35 40 45
Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile
50 55 60
Arg
65
<210> 55
<211> 166
<212> PRT
<213> Artificial Sequence
<220>
<223> IFNβ
<400> 55
Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln
1 5 10 15
Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu
20 25 30
Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln
35 40 45
Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln
50 55 60
Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn
65 70 75 80
Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn
85 90 95
His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr
100 105 110
Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg
115 120 125
Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr
130 135 140
Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu
145 150 155 160
Thr Gly Tyr Leu Arg Asn
165
<210> 56
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Panitumumab (H)-Fc1
<400> 56
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly
20 25 30
Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu
35 40 45
Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr
85 90 95
Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly
100 105 110
Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
355 360 365
Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 57
<211> 573
<212> PRT
<213> Artificial Sequence
<220>
<223> Panitumumab(L)-(GGGGS)3-SIRPαD1-Fc2
<400> 57
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu
85 90 95
Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys
225 230 235 240
Ser Val Ser Val Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val
245 250 255
Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly
260 265 270
Pro Ala Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg
275 280 285
Val Thr Thr Val Ser Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser
290 295 300
Ile Ser Ile Ser Ala Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys
305 310 315 320
Val Lys Phe Arg Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala
325 330 335
Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Asp Lys Thr His Thr Cys
340 345 350
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
355 360 365
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
370 375 380
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
385 390 395 400
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
405 410 415
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
420 425 430
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
435 440 445
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
450 455 460
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
465 470 475 480
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
485 490 495
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
500 505 510
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
515 520 525
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
530 535 540
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
545 550 555 560
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 58
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Tacatuzumab(H)-Fc1
<400> 58
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr
20 25 30
Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Tyr Trp Ile
35 40 45
Gly Tyr Ile His Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys
85 90 95
Ala Arg Ser Gly Gly Gly Asp Pro Phe Ala Tyr Trp Gly Gln Gly Ser
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys
355 360 365
Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 59
<211> 620
<212> PRT
<213> Artificial Sequence
<220>
<223> Tacatuzumab(L)-(GGGGS)3-IFNα 2b-Fc2
<400> 59
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr
20 25 30
Ile Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Met
35 40 45
His Tyr Thr Ser Ala Leu Leu Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg
225 230 235 240
Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser
245 250 255
Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly
260 265 270
Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile
275 280 285
Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
290 295 300
Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu
305 310 315 320
Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr
325 330 335
Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
340 345 350
Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
355 360 365
Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
370 375 380
Leu Gln Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro
385 390 395 400
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
405 410 415
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
420 425 430
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
435 440 445
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
450 455 460
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
465 470 475 480
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
485 490 495
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
500 505 510
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
515 520 525
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
530 535 540
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
545 550 555 560
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
565 570 575
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
580 585 590
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
595 600 605
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 60
<211> 621
<212> PRT
<213> Artificial Sequence
<220>
<223> Tacatuzumab(L)-(GGGGS)3-IFNβ-Fc2
<400> 60
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr
20 25 30
Ile Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Met
35 40 45
His Tyr Thr Ser Ala Leu Leu Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser
225 230 235 240
Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu
245 250 255
Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile
260 265 270
Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr
275 280 285
Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser
290 295 300
Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr
305 310 315 320
His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys
325 330 335
Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg
340 345 350
Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His
355 360 365
Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe
370 375 380
Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn Asp Lys Thr His Thr Cys
385 390 395 400
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
405 410 415
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
420 425 430
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
435 440 445
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
450 455 460
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
465 470 475 480
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
485 490 495
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
500 505 510
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
515 520 525
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys
530 535 540
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
545 550 555 560
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
565 570 575
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
580 585 590
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
595 600 605
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615 620
<210> 61
<211> 581
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofatumumab(H)-(GGGS)3-SIRPαD1-Fc1
<400> 61
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Glu Glu
225 230 235 240
Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly Glu
245 250 255
Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly Pro
260 265 270
Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr Asn
275 280 285
Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser Thr
290 295 300
Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala Ile Thr Pro
305 310 315 320
Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro
325 330 335
Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala
340 345 350
Lys Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly Lys
580
<210> 62
<211> 441
<212> PRT
<213> Artificial Sequence
<220>
<223> Ofatumumab(L)-Fc2
<400> 62
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
210 215 220
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
225 230 235 240
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
245 250 255
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
260 265 270
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
275 280 285
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
290 295 300
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
305 310 315 320
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
325 330 335
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
340 345 350
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
355 360 365
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
370 375 380
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
385 390 395 400
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
405 410 415
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
420 425 430
Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 63
<211> 456
<212> PRT
<213> Artificial Sequence
<220>
<223> Tiragolumab(H)-Fc
<400> 63
Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Lys Thr Tyr Tyr Arg Phe Lys Trp Tyr Ser Asp Tyr Ala
50 55 60
Val Ser Val Lys Gly Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Phe Tyr Cys Thr Arg Glu Ser Thr Thr Tyr Asp Leu Leu Ala Gly Pro
100 105 110
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
115 120 125
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
130 135 140
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
165 170 175
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
180 185 190
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
210 215 220
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
225 230 235 240
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
290 295 300
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
305 310 315 320
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
340 345 350
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
355 360 365
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
385 390 395 400
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 64
<211> 220
<212> PRT
<213> Artificial Sequence
<220>
<223> Tiragolumab (L)
<400> 64
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220

Claims (28)

1.一种双特异性重组蛋白，其特征在于，所述双特异性重组蛋白包括第一功能结合片段、第二功能结合片段和Fc区；所述双特异性重组蛋白靶向目的抗原的第一功能结合片段包含抗原结合片段，其中抗原结合片段中CL结构域的C末端或CH1结构域的C末端与具有免疫调节功能和/或具有代谢调节功能和/或具有内分泌调节功能的第二功能结合片段直接连接或通过接头序列连接；
优选地，所述抗原结合片段与所述第二功能结合片段的N端直接连接或通过接头序列连接，并且，所述第二功能结合片段的C端直接连接或通过接头序列连接Fc的N端。
2.如权利要求1所述的双特异性重组蛋白，其特征在于，所述具有免疫调节功能的第二功能结合片段靶向免疫检查点、免疫检查点配体或细胞因子受体。
3.如权利要求2所述的双特异性重组蛋白，其特征在于，所述具有免疫调节功能的第二功能结合片段靶向PD-1或其配体、CD47或其配体、CD24或其配体、干扰素受体、白介素受体。
4.如权利要求1所述的双特异性重组蛋白，其特征在于，所述具有代谢调节功能的第二功能结合片段靶向代谢调节因子、代谢调节因子受体。
5.如权利要求4所述的双特异性重组蛋白，其特征在于，所述具有代谢调节功能的第二功能结合片段靶向胰岛素受体、成纤维细胞生长因子受体。
6.如权利要求1所述的双特异性重组蛋白，其特征在于，所述具有内分泌调节功能的第二功能结合片段靶向内分泌调节因子、内分泌调节因子受体。
7.如权利要求6所述的双特异性重组蛋白，其特征在于，所述具有内分泌调节功能的第二功能结合片段靶向激素受体。
8.如权利要求1所述的双特异性重组蛋白，其特征在于，所述抗原结合片段中可变区和恒定区直接连接或通过接头序列连接；或所述抗原结合片段与Fc区直接连接或通过接头序列连接；或同时使用上述两种方式连接。
9.如权利要求1或8所述的双特异性重组蛋白，其特征在于，所述接头序列包含(GGGGS)n、(GGGS)n、(GGS)n、(G)n、(GS)n、(EAAAK)n、或(XP)n，n为自然数；
较佳地，所述n为0-5的自然数。
10.如权利要求2或3所述的双特异性重组蛋白，其特征在于，所述第二功能结合片段结合细胞因子受体或免疫检查点或免疫检查点配体，所述第二功能结合片段为细胞因子或免疫检查点配体或免疫检查点配体结合蛋白，或其功能片段或其突变体；所述第二功能结合片段选自以下任意一种：人源SIRP家族胞外功能片段、人干扰素家族功能片段、肿瘤坏死因子超家族功能片段、TGF-β超家族功能片段、白介素类功能片段、趋化因子家族功能片段、集落刺激因子家族功能片段、生长因子功能片段或其突变体。
11.如权利要求10所述的双特异性重组蛋白，其特征在于，所述第二功能结合片段为人源SIRPα胞外D1结构域或其突变体。
12.如权利要求10所述的双特异性重组蛋白，其特征在于，所述干扰素受体为I型或II型人干扰素受体，优选人干扰素γ(IFN-γ)或人干扰素β(IFN-β)的受体。
13.如权利要求10所述的双特异性重组蛋白，其特征在于，所述第二功能结合片段为白介素、其截短体，或其突变体。
14.如权利要求13所述的双特异性重组蛋白，其特征在于，所述白介素为免疫调节或趋化因子，选自以下任意一种：IL-1家族、IL-2家族、IL-3家族、IL-6家族、IL-8家族、IL-10家族、IL-12家族和IL-17家族。
15.如权利要求1-14任一项所述的双特异性重组蛋白，其特征在于，所述第一功能结合片段靶向选自下列靶标中的任意一种或多种：5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD71、CD74、CD79b、CD80、CD83、CD86、CD98、CD206、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CCR5、CXCR4、DLL-4、EGFR、EGFRvIII、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、Notch 1、PD-1、PD-L1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、TIGIT、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、BCMA、CD138、TROP2、Siglec15、CD155和AFP；
较佳地，所述第二功能结合片段包含人源SIRPα胞外D1结构域或其突变体、人干扰素β或人干扰素γ、白介素、其截短体或突变体，所述第一功能结合片段靶向肿瘤细胞或免疫细胞。
16.如权利要求1-14任一项所述的双特异性重组蛋白，其特征在于，所述双特异性重组蛋白由A链和B链组成，所述A链与B链通过分子间作用力结合，或通过共价键结合，或通过盐键结合，或通过上述结合方式中的两种或三种的组合而结合。
17.如权利要求16所述的双特异性重组蛋白，其特征在于，所述Fc区包含Fc区天然序列或Fc非天然序列。
18.如权利要求17所述的双特异性重组蛋白，其特征在于，所述Fc区为人Fc区；
较佳地，所述A链和B链的Fc区是通过knobs-into-holes结合的；和/或，所述Fc区为人IgG的Fc区；
更佳地，所述Fc区为人IgG1或IgG4的Fc区。
19.如权利要求17或18所述的双特异性重组蛋白，其特征在于，所述CL结构域的C末端或CH1结构域的C末端或第二功能结合片段的C末端与所述Fc区直接连接或通过接头序列连接。
20.如权利要求17或18所述的双特异性重组蛋白，其特征在于，所述第一功能结合片段为靶向选自下列靶标的抗原结合片段中的任意一种或多种：5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD71、CD74、CD79b、CD80、CD83、CD86、CD98、CD206、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CCR5、CXCR4、DLL-4、EGFR、EGFRvIII、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、Notch 1、PD-1、PD-L1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、TIGIT、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、BCMA、CD138、TROP2、Siglec15、CD155和AFP；所述抗原结合片段为人鼠嵌合抗原结合片段、人源化抗原结合片段、或全人源抗原结合片段。
21.如权利要求20所述的双特异性重组蛋白，其特征在于，
所述第一功能结合片段靶向CD20、EGFR、EGFRvIII、PD-L1、PD-L2、HER2、HER3、CD138、CD44、CD24、EpCAM、CLDN18.2、CD38、BCMA、MUC1、或TROP2时，所述第二功能结合片段包含SIRPα胞外D1结构域或其突变体；
所述第一功能结合片段靶向TIGIT、Siglec15、PD-1、PD-L1、PD-L2、CD71、CD80、CD86、CD206、CCR5时，所述第二功能结合片段包含IFN-β、IFN-γ、IL-10M、IL-12A，或IL-15和IL15RαSUSHI形成的复合体，或其突变体；
较佳地：
所述第一功能结合片段靶向CD20、EpCAM、CD24或EGFR，所述第二功能结合片段包含SIRPα胞外D1结构域或其突变体；优选所述第二功能结合片段的氨基酸序列如SEQ ID NO:50所示；更优选所述A链的氨基酸序列如SEQ ID NO:1所示，所述B链的氨基酸序列如SEQ IDNO:2或3所示；所述A链的氨基酸序列如SEQ ID NO:61所示，所述B链的氨基酸序列如SEQ IDNO:62所示；所述A链的氨基酸序列如SEQ ID NO:27所示，所述B链的氨基酸序列如SEQ IDNO:28所示；所述A链的氨基酸序列如SEQ ID NO:29所示，所述B链的氨基酸序列如SEQ IDNO:30所示；所述A链的氨基酸序列如SEQ ID NO:56所示，所述B链的氨基酸序列如SEQ IDNO:57所示；
所述第一功能结合片段靶向TIGIT、CD80或PD-1，所述第二功能结合片段包含IL-12A、IL-10单体突变体、IL15与IL-15RαSUSHI复合体，所述IL-12A、IL-10单体突变体、IL15与IL-15RαSUSHI的氨基酸序列分别如SEQ ID NO:51、52、53、54所示，或上述第二功能结合片段的突变体；优选所述A链的氨基酸序列如SEQ ID NO:35所示，所述B链的氨基酸序列如SEQ IDNO:36所示；所述A链的氨基酸序列如SEQ ID NO:37所示，所述B链的氨基酸序列如SEQ IDNO:38所示；所述A链的氨基酸序列如SEQ ID NO:39所示，所述B链的氨基酸序列如SEQ IDNO:40或41所示；
所述第一功能结合片段靶向CD38或AFP，所述第二功能结合片段包含SIRPα胞外D1结构域或其突变体、或者IFN-β或其突变体；优选所述第二功能结合片段的氨基酸序列如SEQ IDNO:50或55所示；更优选所述A链的氨基酸序列如SEQ ID NO:31所示，所述B链的氨基酸序列如SEQ ID NO:32所示；所述A链的氨基酸序列如SEQ ID NO:33所示，所述B链的氨基酸序列如SEQ ID NO:34所示；或，所述A链的氨基酸序列如SEQ ID NO:58所示，所述B链的氨基酸序列如SEQ ID NO:60所示。
22.编码如权利要求1-21任一项所述的双特异性重组蛋白的核酸分子；其特征在于，其中编码所述第一功能结合片段的核酸分子与编码第二功能结合片段的核酸在同一条DNA链中，或编码所述第一功能结合片段的核酸分子与编码第二功能结合片段的核酸在不同的DNA链中；
较佳地，编码所述Fc区的核酸分子与编码所述第一功能结合片段或第二功能结合片段的核酸在同一条DNA链中，编码所述Fc区的核酸分子与编码所述第一功能结合片段或第二功能结合片段的核酸在不同的DNA链中。
23.包含如权利要求22所述的核酸分子的表达载体。
24.由如权利要求23所述的表达载体转化而成的宿主细胞。
25.一种如权利要求1-21任一项所述的双特异性重组蛋白的制备方法，其使用如权利要求23所述的表达载体转化成如权利要求24所述的宿主细胞，在适合表达的条件下培养上述宿主细胞，表达即得所述双特异性重组蛋白。
26.一种药物或药物组合物，其包含如权利要求1-21任一项所述的双特异性重组蛋白。
27.如权利要求1-21任一项所述的双特异性重组蛋白在制备治疗肿瘤、自身免疫性疾病、感染疾病、败血症、移植物抗宿主病、代谢紊乱、内分泌紊乱的药物中的应用。
28.如权利要求27所述的应用，其特征在于，所述肿瘤为实体瘤或血液瘤；优选地，所述实体瘤选自以下任意一种：乳腺癌、结直肠癌、肺癌、胰腺癌、食管癌、子宫内膜癌、卵巢癌、胃癌、前列腺癌、肾癌、宫颈癌、甲状腺癌、子宫癌、膀胱癌、神经内分泌癌、头颈癌、肝癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、皮肤纤维肉瘤突出症、梅克尔细胞癌、胶质母细胞瘤、神经胶质瘤、肉瘤、间皮瘤或骨髓发育不良综合征；所述血液瘤选自骨髓瘤、淋巴瘤或白血病；
所述自身免疫性疾病选自以下任意一种：桥本氏甲状腺炎、1型糖尿病、系统性红斑狼疮、类风湿性关节炎、干燥综合征；
所述感染疾病选自以下任意一种：病毒性感染、细菌感染、真菌感染和其他病原体感染。
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