CN113286822A - 靶向肿瘤的超激动性cd28抗原结合分子 - Google Patents
靶向肿瘤的超激动性cd28抗原结合分子 Download PDFInfo
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- C07K2317/00—Immunoglobulins specific features
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- C07K2317/71—Decreased effector function due to an Fc-modification
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- C—CHEMISTRY; METALLURGY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/75—Agonist effect on antigen
Abstract
本发明涉及能够与CD28多价结合的靶向肿瘤的超激动性抗原结合分子、其生产方法、含有这些抗体的药物组合物、及其使用方法。
Description
技术领域
本发明涉及靶向肿瘤的超激动性CD28抗原结合分子、其生产方法、含有这些分子的药物组合物、及其在癌症治疗中作为免疫调节剂的用途。
背景技术
癌症免疫疗法正在成为一种越来越有效的疗法选择,它可以在诸如黑色素瘤、非小细胞肺癌和肾细胞癌等癌症类型中产生显著而持久的响应。这主要是由几种免疫检查点阻断药物的成功推动的,这些药物包括抗PD-1(例如,Keytruda,Merck;Opdivo,BMS)、抗CTLA-4(例如,Yervoy,BMS)和抗PD-L1(例如,Tecentriq,Roche)。这些药物很可能充当许多癌症类型的护理标准,或作为联合疗法的基础,但是,只有一小部分患者(<25%)从此类疗法中受益。此外,各种癌症(前列腺癌、结直肠癌、胰腺癌、肉瘤、非三阴性乳腺癌等)对这些免疫调节剂表现出原发性耐药性。许多报告表明,缺乏预先存在的抗肿瘤T细胞会导致一些患者的响应缺乏或不佳。总而言之,尽管现有免疫疗法具有令人印象深刻的抗癌作用,但是,对于解决大量的癌症患者群体和对于开发旨在诱导并增强新型肿瘤特异性T细胞响应的疗法,仍然有明确的医学需要。
CD28是共刺激分子亚家族的创始成员,其特征是成对的V-set免疫球蛋白超家族(IgSF)结构域附接至单个跨膜结构域和包含关键信号传导基序的胞质结构域(Carreno和Collins,2002)。该亚家族的其他成员包括ICOS、CTLA-4、PD1、PD1H、TIGIT和BTLA(Chen和Flies,2013)。CD28的表达仅限于T细胞,并普遍存在于所有幼稚的亚组和大多数接触过抗原的亚组中,包括那些表达PD-1或CTLA-4的亚组。CD28和CTLA-4具有高度同源性,并竞争与表达在树突细胞、B细胞、巨噬细胞和肿瘤细胞上的相同B7分子CD80和CD86的结合(Linsley等人,1990)。CTLA-4对B7家族配体的较高亲和力使CTLA-4在配体结合方面胜过CD28并抑制效应T细胞响应(Engelhardt等人,2006)。相比之下,PD-1被证明通过部分地去磷酸化CD28的胞质结构域来抑制CD28信号传导(Hui等人,2017)。对于幼稚T细胞的功能性从头引发、随后的克隆扩增、细胞因子产生、靶细胞裂解和长期记忆的形成,严格要求通过专职性抗原呈递细胞表面上的CD80或CD86进行CD28的连接。CD28配体的结合还促进了诱导型共刺激受体诸如OX-40、ICOS和4-1BB的表达(在Acuto和Michel,2003中综述)。
已经证明,在CD28连接后,二硫键连接的同型二聚体,即,膜近端YMNM基序和远端PYAP基序与几种激酶和衔接蛋白复合(Boomer和Green,2010)。这些基序对于诱导IL2转录很重要,IL2转录是由NFAT、AP-1和NFκB家族转录因子的CD28依赖性活化介导的(Fraser等人,1991)(June等人,1987)(Thompson等人,1989)。但是,在CD28的胞质结构域中发现了其他特征较弱的磷酸化和泛素化位点。
如(Esensten等人,2016)所综述,CD28起始的途径在促进常规T细胞的增殖和效应子功能中起关键作用。CD28连接还可以促进调节性T细胞的抗炎功能。CD28通过部分地增强来自T细胞受体的信号而共刺激T细胞,但也被证明介导独特的信号传导事件(Acuto和Michel,2003;Boomer和Green,2010;June等人,1987)。由CD28特异性触发的信号控制T细胞功能的许多重要方面,包括下游蛋白质的磷酸化和其他翻译后修饰(例如,PI3K介导的磷酸化)、转录变化(例如,Bcl-xL表达)、表观遗传变化(例如,IL-2启动子)、细胞骨架重塑(例如,微管组织中心的取向)和糖酵解速率的变化(例如,糖酵解通量)。
CD28缺陷型小鼠对感染性病原体、同种异体移植抗原、移植物抗宿主病、接触性超敏反应和哮喘的响应降低(Acuto和Michel,2003)。缺乏CD28介导的共刺激会导致体外和体内T细胞增殖减少,严重抑制生发中心形成和免疫球蛋白同种型转换、减少辅助性T(Th)细胞分化和Th2型细胞因子的表达。CD4依赖性细胞毒性CD8+T细胞响应也受到影响。重要的是,CD28缺陷性幼稚T细胞显示出减少的增殖性响应,尤其是在较低的抗原浓度下。
越来越多的文献支持将CD28结合在T细胞上具有抗肿瘤潜能的观点。最近的证据表明,PD-L1/PD-1和CTLA-4检查点抑制剂的抗癌作用取决于CD28(Kamphorst等人,2017;Tai等人,2007)。研究CTLA-4和PD-1阻断药物的治疗效果的临床研究已经显示,对晚期黑色素瘤和其他癌症患者的治疗结果异常乐观。此外,输注表达人工嵌合T细胞受体(包括与细胞内TCR信号传导结构域(CD3z)和细胞内共刺激结构域(CD28和/或4-1BB结构域)融合的细胞外抗原识别结构域)的经基因工程化改造的T细胞,在B细胞癌和其他癌症中表现出高响应率和持久性。
CD28激动性抗体可以分为两类:(i)CD28超激动性抗体和(ii)CD28常规激动性抗体。通常,为了活化幼稚的T细胞,既需要T细胞抗原受体(TCR,信号1)的参与,也需要CD28的共刺激信号传导(信号2)。CD28超激动剂(CD28SA)是CD28特异性的单克隆抗体,其能够自主活化T细胞,而没有明显的T细胞受体参与(Hunig,2012)。在啮齿动物中,CD28SA活化常规和调节性T细胞。CD28SA抗体在多种自身免疫、炎症和移植模型中具有治疗效果。但是,在2006年,人CD28SA抗体TGN1412的I期研究引起了威胁生命的细胞因子风暴。后续研究表明,毒性是由于人T细胞与临床前动物模型的T细胞对CD28的响应性不同而导致的剂量错误引起的。目前,正在针对RA患者和患有转移性或不可切除的晚期实体恶性肿瘤的患者的开放标签、多中心剂量递增研究中重新评估TGN1412。CD28常规激动性抗体(例如,克隆体9.3)模仿CD28天然配体,并且仅在存在T细胞受体信号的情况下才能增强T细胞活化。已发表的见解表明,抗体的结合表位对激动性抗体是超激动剂还是常规激动剂具有重大影响(Beyersdorf et al.,2005)。超激动性TGN1412与CD28的侧基序结合,而常规激动性分子9.3与配体结合表位紧密结合。由于结合表位的不同,超激动性抗体和常规激动性抗体在T细胞表面形成CD28分子线性复合物的能力也不同。准确地说,TGN1412能够有效地形成CD28的线性阵列,这大概导致聚集的信号成分足以超过T细胞活化的阈值。另一方面,常规激动剂9.3导致结构不是线性的复合物。先前已经公开了使用针对黑色素瘤相关蛋白聚糖和CD28的重组双特异性单链抗体来转换基于克隆体9.3的传统激动性结合剂的尝试(Otz etal.,2009)。基于双特异性单链抗体形成多聚性构建体的内在趋势,尽管使用了常规的CD28激动性结合剂9.3,但已报道的双特异性单链抗体仍具有“超激动”活性。然而,这些构建体依赖于稳定和一致的多聚化。
发明内容
本发明描述了靶向肿瘤的超激动性CD28抗原结合分子,其实现了肿瘤依赖性的自主T细胞活化和肿瘤细胞杀死,而无需形成多聚体。这些CD28抗原结合分子的特征在于它们能够与CD28多价结合,并且它们包含至少一个能够与肿瘤相关抗原诸如成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)特异性结合的抗原结合结构域。此外,它们具有由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代。从而消除了Fc受体介导的交联,并且通过至少一种能够特异性结合肿瘤相关抗原的抗原结合域与其抗原的结合来交联,从而实现了肿瘤特异性活化。
因此,本发明提供了超激动性CD28抗原结合分子,其能够与CD28多价结合并且包含
(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,
(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,和
(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代。
因此,本发明涉及能够在不事先活化T细胞的情况下诱导T细胞增殖和细胞因子分泌的CD28抗原结合分子。但是,仅当它与肿瘤相关抗原结合时,它才会诱导T细胞增殖和细胞因子分泌而无需事先活化T细胞,这是由于CD28抗原结合分子缺少Fc受体和/或效应子功能,因此需要通过至少一种能够特异性结合至肿瘤相关抗原的抗原结合结构域与其抗原结合而交联。
在一方面,提供了如下定义的超激动性CD28抗原结合分子,其中Fc结构域是IgG,特别是IgG1 Fc结构域或IgG4 Fc结构域。在一个特定方面,由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域是IgG1 Fc结构域。在一方面,Fc结构域包含氨基酸取代L234A和L235A(根据Kabat的EU索引编号)。在一方面,Fc结构域属于人IgG1亚类并且包含氨基酸突变L234A、L235A和P329G(根据Kabat EU索引编号)。
在一方面,提供了如前文所定义的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(i)重链可变区(VHCD28),其包含SEQ ID NO:20的重链互补决定区CDR-H1、SEQ IDNO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的轻链互补决定区CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3;或
(ii)重链可变区(VHCD28),其包含SEQ ID NO:36的CDR-H1、SEQ ID NO:37的CDR-H2和SEQ ID NO:38的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:39的CDR-L1、SEQ ID NO:40的CDR-L2和SEQ ID NO:41的CDR-L3。
在一方面,超激动性CD28抗原结合分子的能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:36的CDR-H1、SEQ ID NO:37的CDR-H2和SEQ ID NO:38的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:39的CDR-L1、SEQ ID NO:40的CDR-L2和SEQ ID NO:41的CDR-L3。
在另一方面,超激动性CD28抗原结合分子的能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:20的CDR-H1、SEQ ID NO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3。
此外,提供了如前文所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含与SEQ ID NO:26的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCD28),其包含与SEQ ID NO:27的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
在进一步方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含选自由SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51组成的组的氨基酸序列;以及轻链可变区(VLCD28),其包含选自由SEQ ID NO:27、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61组成的组的氨基酸序列。
在另一方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(a)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(b)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(c)重链可变区(VHCD28),其包含SEQ ID NO:51的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:61的氨基酸序列,或
(d)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(e)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(f)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(g)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(h)重链可变区(VHCD28),其包含SEQ ID NO:43的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(i)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(j)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(k)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列。
在一个特定方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列;以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列。
在另一个特定方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列;以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列。
在进一步特定方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列;以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列。
在进一步方面,提供了如前文所定义的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均为Fab片段。
在一方面,提供了超激动性CD28抗原结合分子,其中能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与癌胚抗原(CEA)特异性结合的抗原结合结构域。
在一方面,提供了如本文所述的超激动性CD28抗原结合分子,其中能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含:(i)CDR-H1,其包含SEQ IDNO:127的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:128的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:129的氨基酸序列;以及轻链可变区(VLCEA),其包含:(iv)CDR-L1,其包含SEQ ID NO:130的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:131的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:132的氨基酸序列。特别地,能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含与SEQ ID NO:133的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCEA),其包含与SEQ IDNO:134的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
在另一方面,提供了超激动性CD28抗原结合分子,其中能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与成纤维细胞活化蛋白(FAP)特异性结合的抗原结合结构域。
在一方面,提供了如本文所述的超激动性CD28抗原结合分子,其中能够与FAP特异性结合的抗原结合结构域包含
(a)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:12的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:13的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:14的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:15的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:16的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:17的氨基酸序列;或
(b)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:4的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:5的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:6的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:7的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:8的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:9的氨基酸序列。特别地,能够与FAP特异性结合的抗原结合结构域包含:重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:12的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:13的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:14的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:15的氨基酸序列,(v)CDR-L2,其包含SEQID NO:16的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:17的氨基酸序列。
在一方面,提供了超激动性CD28抗原结合分子,其中能够与FAP特异性结合的抗原结合结构域包含:(a)重链可变区(VHFAP),其包含与SEQ ID NO:18的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ ID NO:19的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;或者(b)重链可变区(VHFAP),其包含与SEQ ID NO:10的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ ID NO:11的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列。特别地,能够与FAP特异性结合的抗原结合结构域包含:重链可变区(VHFAP),其包含SEQ ID NO:18的氨基酸序列;以及轻链可变区(VLFAP),其包含SEQ ID NO:19的氨基酸序列。
在另一方面,提供了如本文所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的VH和VL结构域,其中所述VH结构域经由肽连接基与两条重链中的一条的C末端联结,并且其中所述VL结构域经由肽连接基与第二重链的C末端联结。
在进一步方面,提供了如本文所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的crossFab片段,其经由肽连接基与两条重链中的一条的C末端联结。
在另一方面,提供了如本文所公开的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的两个crossFab片段,其中一个crossFab片段经由肽连接基与两条重链中的一条的C末端联结,并且其中另一个crossFab片段经由肽连接基与第二重链的C末端联结。
根据本发明的另一方面,提供了编码本发明的抗体或双特异性抗原结合分子的分离的多核苷酸。本发明进一步提供了一种或多种表达载体,其包含本发明的分离的多核苷酸;并且提供了宿主细胞,其包含本发明的分离的多核苷酸或表达载体。在一些方面,宿主细胞是真核细胞,特别是哺乳动物细胞。在另一方面,提供了生产如本文所述的超激动性CD28抗原结合分子的方法,其包括在适合于表达该超激动性CD28抗原结合分子的条件下培养本发明的宿主细胞。任选地,该方法还包括回收超激动性CD28抗原结合分子。本发明还涵盖通过本发明的方法生产的超激动性CD28抗原结合分子。
本发明进一步提供了药物组合物,其包含本发明的超激动性CD28抗原结合分子以及至少一种药用赋形剂。在一个方面,药物组合物用于治疗癌症。
本发明还涵盖使用本发明的超激动性CD28抗原结合分子和药物组合物的方法。在一方面,本发明提供了根据本发明的超激动性CD28抗原结合分子或药物组合物,其用作药物。在一方面,提供了根据本发明的超激动性CD28抗原结合分子或药物组合物,其用于治疗疾病。在一个具体方面,疾病是癌症。在另一方面,提供了根据本发明的超激动性CD28抗原结合分子或药物组合物,其用于治疗癌症,其中超激动性CD28抗原结合分子与化疗剂、放疗和/或用于癌症免疫疗法的其他药剂联合施用。
还提供了根据本发明的超激动性CD28抗原结合分子在制备用于治疗疾病的药物中的用途;以及在个体中治疗疾病的方法,其包括向所述个体施用治疗有效量的药用形式的根据本发明的超激动性CD28抗原结合分子或包含根据本发明的超激动性CD28抗原结合分子的组合物。在一个具体方面,疾病是癌症。在另一方面,提供了根据本发明的超激动性CD28抗原结合分子在制备用于治疗疾病的药物中的用途,其中治疗包括与化疗剂、放疗和/或其他用于癌症免疫疗法的药剂联合施用。在进一步方面,提供了治疗个体中的疾病的方法,其包括向所述个体施用治疗有效量的药用形式的根据本发明的超激动性CD28抗原结合分子或包含根据本发明的超激动性CD28抗原结合分子的组合物,其中该方法包括与化疗剂、放疗和/或其他用于癌症免疫疗法的药剂联合施用。还提供了抑制个体中肿瘤细胞生长的方法,其包括向个体施用有效量的药用形式的根据本发明的超激动性CD28抗原结合分子或包含根据本发明的超激动性CD28抗原结合分子的组合物,以抑制肿瘤细胞的生长。在上述方面中的任何方面,个体优选为哺乳动物,特别是人。
附图说明
在图1A至1L中,显示了所述分子的示意图。图1A显示了其huIgG4同种型(TGN1412)形式的CD28激动性抗体CD28(SA)。
图1B显示了作为hu IgG1 PGLALA同种型(“Fc沉默”)的CD28(SA)激动性抗体。
图1C、1D、1E和1F中分别显示了1+1格式、1+2格式、2+2格式和1+4格式的双特异性FAP-CD28抗原结合分子。
图1G、1H和1J中分别显示了1+2格式、2+2格式和1+1格式的双特异性CEA-CD28抗原结合分子。
图1I显示了作为单价hu IgG1 PGLALA同种型(“Fc沉默”)的CD28激动性抗体变体的示意图。
1+1+2格式的三特异性CEA-FAP-CD28抗原结合分子分别以两种替代格式显示在图1K和1L中。
图2A、2B、2C、2D和2E涉及CD28激动性抗体和FAP-CD28抗原结合分子与细胞上的人CD28或人FAP的结合。在图2A中显示了IgG4同种型的CD28(SA)和hu IgG1 PGLALA同种型与人CD28的结合,并且显示了不同FAP-CD28分子与细胞上的人CD28(图2B)和人FAP(图2C)的结合。通过流式细胞术评估不同CD28激动性抗体或抗DP47靶向分子与表达人CD28的CHO细胞(亲代细胞系CHO-k1 ATCC#CCL-61,经修饰以稳定地过表达人CD28)或表达人FAP的3T3细胞(NIH/3T3细胞系(ATCC CRL-1658))结合的中位荧光强度。使用SEM对三种技术性平行样品进行描述。FAP(4B9)-CD28(SA)抗原结合分子(实例1中所述的分子D、E和F)的比较显示在图2D(与人CD28结合)和图2E(与人FAP结合)中。
CD28(SA)的可变结构域及其变体的比对显示在图3A至3D中。为了去除半胱氨酸50并将所得抗CD28结合剂的亲和力降低到不同程度,CD28(SA)VH结构域及其变体的比对显示在图3A和3B中。值得注意的是,在VH变体i和j中,CD28(SA)的CDR从IGHV1-2框架移植到IGHV3-23框架中(图3B)。在图3C和3D中,显示了为了将所得抗CD28结合剂的亲和力降低到不同程度,CD28(SA)VL结构域及其变体的比对。在变体t中,CDR被移植到曲妥珠单抗(赫赛汀)VL序列的框架序列中。
在图4A至4C中,显示了来自上清液的单价IgG形式的亲和力降低的CD28激动性抗体变体与细胞上的人CD28的结合。通过流式细胞术评估与表达人CD28的CHO细胞(亲代细胞系CHO-k1 ATCC#CCL-61,经修饰以稳定地过表达人CD28)的结合的中位荧光强度,与阴性对照(抗DP47)和原始TGN1412进行比较。变体1至10的结合曲线显示在图4A中,变体11至22的结合曲线显示在图4B中,而变体23至31的结合曲线显示在图4C中。使用SD对两种技术性平行样品进行描述。
在图4D和4E中,显示了具有所选择的亲和力降低的CD28激动性抗体变体的huIgG1PG-LALA 1+1格式的靶向FAP的双特异性CD28激动性抗体变体与细胞上的人CD28的结合。具有变体8、11、12、15、16和17的双特异性1+1构建体的结合曲线显示在图4D中,而具有变体19、23、25、27和29的双特异性1+1构建体的结合曲线显示在图4E中。基于亲和力来选择所选择的结合剂用于生产1+1、双特异性靶向FAP的格式。显示了通过流式细胞术评估的与表达人CD28的CHO细胞(亲代细胞系CHO-k1 ATCC#CCL-61,经修饰以稳定地过表达人CD28)的结合的中位荧光强度,与阴性对照(抗DP47)和原始TGN1412(分子A)进行比较。
所选择的huIgG1 PG-LALA 1+1格式的靶向FAP的双特异性CD28激动性抗体的体外效力在图4F和4G中示出。在MCSP-TCB(5pM,P1AD2189)浓度受限而FAP-CD28构建体浓度递增的情况下,用表达MCSP和FAP的MV3黑色素瘤细胞将T细胞孵育5天。在图4F中显示了CFSE稀释度,其作为通过流式细胞术评估的CD8 T细胞的T细胞增殖的量度。误差线显示SEM,图描述了来自2个供体的代表性结果的三次技术性平行实验。在图4G中显示了CD28结合剂变体的KD(nM)与以曲线下面积(作为占亲代TGN1412克隆体(CD28(SA))的百分比)表示的效力(a)的相关性。
图5A至5D涉及CD28(SA)的高密度(HD)预培养的建立和作用方式。将PBMC T细胞以高密度(HD)预培养2天,或从PBMC中新鲜分离而使用,并且用递增浓度的CD28(SA)刺激。描绘了CFSE稀释度,作为在使用CD28(SA)(分子A,P1AE1975)刺激之后5天后的T细胞增殖的指标(图5A)和刺激之后2天后的细胞因子分泌的指标(图5B)。图5C显示了在HD PBMC预培养之前和2天之后,通过流式细胞术评估的PBMC单核细胞和B细胞中FcγRIIb表达的百分比。图5D:在存在或不存在FcγRIIb阻断抗体或同种型对照的情况下,将HD预培养的PBMC与CD28(SA)共培养5天,并通过流式细胞术评估CD4 T细胞的CFSE稀释百分比。图代表至少6个供体(图5A、5B)和2个供体(图5C、5D),每个都在独立实验中评估。这些图显示了平行实验。误差线表示SEM。统计分析是通过学生t检验进行的。***:p<0.001。CD28(SA)IgG4的超激动性取决于与FcγRIIb的交联。
在图6A和6B中,T细胞增殖,即,用原始Fc野生型IgG4 CD28(SA)(P1AE1975)或带有P329G-LALA突变的CD28(SA)(P1AD9289)刺激5天后CD4 T细胞的CFSE稀释度。T细胞以高密度预培养2天。图代表至少3个独立实验。显示了三次技术性平行实验。Fc沉默消除了TGN1412中的超激动作用。向Fc沉默的TGN1412添加肿瘤靶向部分恢复了超激动性,然后,超激动性取决于肿瘤靶的存在。
在图7A、7B、7C和图7D中,显示了具有超激动性(CD28(SA))结合剂和常规激动性结合剂(9.3,CD28(CA))的不同形式(2+2和1+2)的靶向FAP的CD28激动剂的比较。具有常规CD28激动性结合剂的靶向FAP的CD28激动剂不能用作超激动剂。在存在递增浓度的具有超激动性结合剂(SA,图7A)或常规激动性结合剂(9.3,图7B)的FAP-CD28格式的情况下,将PBMC T细胞与3T3-huFAP细胞(FAP存在)共培养5天。显示了T细胞增殖。然后,还在存在递增浓度的具有超激动性结合剂(SA,图7C)或常规激动性结合剂(9.3,图7D)的FAP-CD28格式的情况下,将PBMC T细胞与3T3 WT细胞(FAP不存在)共培养5天。描述了作为CD8 T细胞的T细胞增殖的量度的CFSE稀释度,在刺激后第5天通过流式细胞术评估。图显示了3个独立实验中3个供体的累积数据。误差线显示SEM。在相同的实验设置中,共培养2天后,还从上清液中测量了细胞因子。结果提供在图7E中。
在90小时的进程中,使用IncuCyte技术通过活细胞成像评估了具有超激动性CD28(SA)结合剂或常规激动性结合剂(CD28(CA))的各种格式的FAP-CD28诱导杀死表达FAP的RFP-MV3黑色素瘤细胞的能力。包括FAP-TCB(P1AD4645)在内的所有分子均以10nM使用。图8A、8B和图8C分别显示了来自三个供体的三次技术性平行实验的代表性结果。图8D显示了来自3个独立实验的3个供体在t=90h时的曲线下面积(AUC)表示的累积结果。方框显示第25至第75百分位,须显示最小到最大。通过成对的单因素方差分析进行统计分析。***:p<0.001,ns:不显著。
图9A和9B显示了具有超激动性结合剂和常规激动性结合剂的不同形式的靶向CEA的CD28激动剂的比较。在90小时的进程中,使用IncuCyte技术通过活细胞成像评估了具有超激动性CD28(SA)结合剂或常规激动性结合剂(CD28(CA))的各种格式的CEA-CD28诱导杀死表达CEA的RFP+MKN45胃癌细胞的能力。包括CEACAM5-TCB(P1AD5299)在内的所有分子均以10nM使用。图9A显示了来自一个供体的三次技术性平行实验的代表性结果。图9B显示了在1个实验中1个供体在t=90h时的曲线下面积(AUC)表示的三次技术性平行实验的统计分析。方框显示第25至第75百分位,须显示最小到最大。通过成对的单因素方差分析进行统计分析。***:p<0.001。结果表明,具有常规CD28激动性结合剂的靶向CEA的CD28激动剂的行为不具有超激动性。
在图10A、10B和10C中,显示了具有单价超激动性结合剂的靶向CD28激动剂不是功能上超激动性的。在存在递增浓度的具有二价CD28结合剂的FAP-CD28(P1AD9011,实心圆)或具有CD28结合单价态的FAP-CD28(P1AD4492,空心圆)的情况下,将PBMC T细胞与3T3-huFAP细胞共培养5天。在图10A中显示了CD8 T细胞的CFSE稀释度。此外,通过用流式细胞术检测活化标志物CD69(图10B)和CD25(图10C)来评估T细胞的活化。在刺激后5天,显示了CD69和CD25染色的平均荧光强度(MFI)。显示了来自1个供体的三次技术性平行实验,误差线表示SEM。表明类似于TGN1412的超激动作用需要多价CD28结合。
具体实施方式
定义
除非另有定义,否则本文使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的,将应用以下定义,并且在适当时,以单数形式使用的术语也将包括复数,反之亦然。
如本文所用,术语“抗原结合分子”在其最广义上是指特异性结合抗原决定簇的分子。抗原结合分子的示例是抗体、多特异性抗体(例如,双特异性抗体)、抗体片段和支架抗原结合蛋白。
如本文所用,术语“与肿瘤相关抗原结合的抗原结合结构域”或“能够与肿瘤相关抗原特异性结合的部分”是指与抗原决定簇特异性结合的多肽分子。在一个方面,抗原结合结构域能够通过其靶细胞抗原活化信号传导。在一个特定方面,抗原结合结构域能够将与其所连接的实体(例如CD28超激动剂)引导至靶位点,例如引导至携带抗原决定簇的特定类型的肿瘤细胞或肿瘤基质。能够特异性结合到靶细胞抗原的抗原结合结构域包括如本文进一步定义的抗体及其片段。另外,能够特异性结合到靶细胞抗原的抗原结合结构域包括如本文进一步定义的支架抗原结合蛋白,例如基于设计的重复序列蛋白或设计的重复序列结构域的结合结构域(参见例如WO 2002/020565)。
关于抗原结合分子即抗体或其片段,术语“与靶细胞抗原特异性结合的抗原结合结构域”是指分子的一部分,其包含与抗原的一部分或全部特异性结合并且与之互补的区域。可通过例如一种或多种抗体可变结构域(也称为抗体可变区)来提供能够特异性抗原结合的抗原结合结构域。具体地,能够特异性抗原结合的抗原结合结构域包括抗体轻链可变区(VL)和抗体重链可变区(VH)。在另一方面,“能够与靶细胞抗原特异性结合的抗原结合结构域”也可以是Fab片段或crossFab片段。
本文的术语“抗体”以最广义使用并且涵盖各种抗体结构,包括但不限于单克隆抗体、多克隆抗体、单特异性和多特异性抗体(例如,双特异性抗体),以及抗体片段,只要它们表现出所需的抗原结合活性即可。
如本文所用的术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体,即,除了可能的变异抗体(例如含有天然存在的突变或在单克隆抗体制剂的生产过程中产生,此类变体通常以少量存在)之外,包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反,单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。
如本文所用,术语“单特异性”抗体表示具有一个或多个结合位点的抗体,每个结合位点与相同抗原的相同表位结合。术语“双特异性”意指抗原结合分子能够特异性结合到至少两种独特的抗原决定簇。通常,双特异性抗原结合分子包含两个抗原结合位点,这两个抗原结合位点中的每个抗原结合位点对不同的抗原决定簇具有特异性。在某些实施例中,双特异性抗原结合分子能够同时结合两种抗原决定簇,特别是在两种独特细胞上或相同细胞上表达的两种抗原决定簇。
本申请中所用的术语“价态”表示在对一种独特抗原决定簇具有特异性的抗原结合分子中存在特定数量的对一种独特抗原决定簇具有特异性的结合位点。因此,术语“二价”、“四价”和“六价”分别表示在抗原结合分子中存在对特定抗原决定簇具有特异性的两个结合位点、四个结合位点和六个结合位点。在本发明的特定方面,根据本发明的双特异性抗原结合分子对特定抗原决定簇可以是单价的,意味着它们对所述抗原决定簇仅具有一个结合位点,或者对特定抗原决定簇可以是二价或四价的,意味着它们对所述抗原决定簇分别具有两个结合位点或四个结合位点。
术语“全长抗体”和“完整抗体”在本文中可互换使用地指代具有与天然抗体结构基本上相似的结构的抗体。“天然抗体”是指具有不同结构的天然免疫球蛋白分子。例如,天然IgG类抗体为约150,000道尔顿的异四聚体糖蛋白,由二硫键键合的两条轻链和两条重链组成。从N末端到C末端,每条重链具有可变区(VH)(也称为可变重链结构域或重链可变结构域),后接三个恒定结构域(CH1、CH2和CH3)(也称为重链恒定区)。类似地,从N末端到C末端,每条轻链具有可变区(VL)(也称为可变轻链结构域或轻链可变结构域),后接轻链恒定结构域(CL)(也称为轻链恒定区)。抗体的重链可以分配为五种类型中的一种,所述五种类型被称为α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM),它们中的一些可以进一步分为亚型,例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。抗体的轻链基于其恒定结构域的氨基酸序列,可以归属于两种类型中的一种,这两种类型称为卡帕(κ)和兰姆达(λ)。
“抗体片段”是指完整抗体以外的分子,其包含完整抗体的一部分,所述部分结合完整抗体所结合的抗原。抗体片段的实例包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2;双体抗体、三体抗体、四体抗体、crossFab片段;线性抗体;单链抗体分子(例如scFv);以及单结构域抗体。关于某些抗体片段的综述,参见Hudson等人,Nat Med 9,129-134(2003)。关于scFv片段的综述,参见例如Plückthun在The harmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,Springer-Verlag,New York,pp.269-315(1994)中所述;还可参见WO 93/16185;以及美国专利5,571,894和5,587,458。对于包含挽救受体结合表位残基且具有延长的体内半衰期的Fab片段和F(ab')2片段的讨论,参见美国专利号5,869,046。双体抗体是具有两个抗原结合位点的抗体片段,该双体抗体可以是二价的或双特异性的,参见例如EP 404,097;WO 1993/01161;Hudson等人,Nat Med 9,129-134(2003);以及Hollinger等人,Proc Natl Acad Sci USA 90,6444-6448(1993)。在Hudson等人,NatMed 9,129-134(2003)中也描述了三体抗体和四体抗体。单结构域抗体为包含抗体的全部或部分重链可变结构域或全部或部分轻链可变结构域的抗体片段。在某些实施例中,单结构域抗体是人单结构域抗体(Domantis,Inc.,Waltham,MA;参见例如美国专利6,248,516B1)。抗体片段可以通过各种技术制备,包括但不限于完整抗体的蛋白水解消化以及由重组宿主细胞(例如大肠杆菌或噬菌体)产生,如本文所述。
木瓜蛋白酶消化完整抗体产生两个称为“Fab”片段的相同的抗原结合片段,每个“Fab”片段含有重链可变结构域和轻链可变结构域以及轻链的恒定结构域和重链的第一恒定结构域(CH1)。因此,如本文所用,术语“Fab片段”是指包含轻链片段以及重链的可变重链(VH)结构域和第一恒定结构域(CH1)的抗体片段,所述轻链片段包含可变轻链(VL)结构域和轻链的恒定结构域(CL)。Fab'片段与Fab片段的不同之处在于Fab'片段在重链CH1结构域的羧基末端添加了一些残基,这些残基包括来自抗体铰链区的一个或多个半胱氨酸。Fab'-SH是Fab'片段,其中恒定结构域的半胱氨酸残基具有游离巯基。胃蛋白酶处理产生F(ab')2片段,其具有两个抗原结合位点(两个Fab片段)和Fc区的一部分。
术语“crossFab片段”或“xFab片段”或“交换型Fab片段”是指这样的Fab片段,其中重链和轻链的可变区或恒定区被交换。交换型Fab分子的两种不同链组成是可能的,并且包含在本发明的双特异性抗体中:在一个方面,Fab重链和轻链的可变区被交换,即交换型Fab分子包含由轻链可变结构域(VL)和重链恒定结构域(CH1)组成的肽链,以及由重链可变结构域(VH)和轻链恒定结构域(CL)组成的肽链。该交换型Fab分子也称为CrossFab(VLVH)。在另一方面,当Fab重链和轻链的恒定区被交换时,交换型Fab分子包含由重链可变结构域(VH)和轻链恒定结构域(CL)组成的肽链,以及由轻链可变结构域(VL)和重链恒定结构域(CH1)组成的肽链。该交换型Fab分子也称为CrossFab(CLCH1)。
“单链Fab片段”或“scFab”是由抗体重链可变结构域(VH)、抗体恒定结构域1(CH1)、抗体轻链可变结构域(VL)、抗体轻链恒定结构域(CL)和连接基组成的多肽,其中所述抗体结构域和所述连接基在N-末端至C-末端方向上具有以下顺序中的一种:a)VH-CH1-连接基-VL-CL,b)VL-CL-连接基-VH-CH1,c)VH-CL-连接基-VL-CH1,或d)VL-CH1-连接基-VH-CL;并且其中所述连接基是至少30个氨基酸,优选是32个至50个氨基酸的多肽。所述单链Fab片段经由CL结构域与CH1结构域之间的天然二硫键而稳定化。此外,这些单链Fab分子可以通过经由插入半胱氨酸残基(例如根据Kabat编号的可变重链中的44位和可变轻链中的100位)产生链间二硫键,而进一步稳定化。
“交换型单链Fab片段”或“x-scFab”是由抗体重链可变结构域(VH)、抗体恒定结构域1(CH1)、抗体轻链可变结构域(VL)、抗体轻链恒定结构域(CL)和连接基组成的多肽,其中所述抗体结构域和所述连接基在N末端至C末端方向上具有以下顺序中的一种:a)VH-CL-连接基-VL-CH1和b)VL-CH1-连接基-VH-CL;其中VH和VL一起形成与抗原特异性结合的抗原结合位点,并且其中所述连接基是至少30个氨基酸的多肽。此外,这些x-scFab分子可以通过经由插入半胱氨酸残基(例如根据Kabat编号的可变重链中的44位和可变轻链中的100位)产生链间二硫键,而进一步稳定化。
“单链可变片段(scFv)”是抗体的重链可变区(VH)和轻链可变区(VL)的融合蛋白,通过10至约25个氨基酸的短连接基肽联结。连接基通常富含甘氨酸以获得柔性,以及富含丝氨酸或苏氨酸以获得溶解度,并且可以将VH的N末端与VL的C末端联结,或反之亦然。尽管去除了恒定区并引入了连接基,但该蛋白保留了原始抗体的特异性。scFv抗体例如描述于Houston,J.S.,Methods in Enzymol.203(1991)46-96)中。另外,抗体片段包含单链多肽,所述单链多肽的特征在于具有VH结构域,即能够与VL结构域一起装配到功能性抗原结合位点;或具有VL结构域的特征,即能够与VH结构域一起装配到功能性抗原结合位点,从而提供全长抗体的抗原结合特性。
“支架抗原结合蛋白”是本领域已知的,例如纤连蛋白和设计的锚蛋白重复序列蛋白(DARPin)已被用作抗原结合结构域的替代支架,参见例如Gebauer和Skerra,Engineeredprotein scaffolds as next-generation antibody therapeutics.Curr Opin ChemBiol 13:245-255(2009)和Stumpp等人,Darpins:A new generation of proteintherapeutics.Drug Discovery Today 13:695-701(2008)。在本发明的一个方面中,支架抗原结合蛋白选自由以下项组成的组:CTLA-4(Evibody)、脂质运载蛋白(Anticalin)、蛋白A衍生的分子(诸如蛋白A的Z结构域(亲和体))、A结构域(Avimer/巨型抗体)、血清转铁蛋白(反式体);设计的锚蛋白重复序列蛋白(DARPin)、抗体轻链或重链的可变结构域(单结构域抗体,sdAb)、抗体重链的可变结构域(纳米抗体,aVH)、VNAR片段、纤连蛋白(AdNectin)、C型凝集素结构域(四连接素);新抗原受体β-内酰胺酶的可变结构域(VNAR片段)、人γ-晶体蛋白或泛素蛋白(Affilin分子);人蛋白酶抑制剂的kunitz型结构域、微型体(诸如来自knottin家族的蛋白质)、肽适体和纤连蛋白(adnectin)。CTLA-4(细胞毒性T淋巴细胞相关抗原4)是主要在CD4+T细胞上表达的CD28家族受体。其细胞外结构域具有可变结构域样Ig折叠。对应于抗体CDR的环可以用异源序列取代,以赋予不同的结合性质。经工程化改造以具有不同结合特异性的CTLA-4分子也称为Evibody(例如US7166697B1)。Evibody与抗体(例如结构域抗体)的分离的可变区大小大致相同。关于进一步的细节,参见Journal ofImmunological Methods 248(1-2),31-45(2001)。脂质运载蛋白是细胞外蛋白质家族,其运输小的疏水性分子,诸如类固醇、胆素、类维生素A和脂质。它们具有刚性β-片层二级结构,在锥形结构的开口端处具有许多环,可以工程化改造成与不同的靶抗原结合。Anticalin的大小介于160-180个氨基酸之间,并且来源于脂质运载蛋白。关于进一步的细节,参见Biochim Biophys Acta 1482:337-350(2000)、US7250297B1和US20070224633。亲和体是来源于金黄色酿脓葡萄球菌(Staphylococcus aureus)的蛋白A的支架,其可以被工程化以结合抗原。该结构域由约58个氨基酸的三螺旋束组成。已经通过表面残基的随机化形成了文库。关于进一步的细节,参见Protein Eng.Des.Sel.2004,17,455-462和EP1641818A1。Avimer是来源于A结构域支架家族的多结构域蛋白质。约35个氨基酸的天然结构域采用确定的二硫键键合结构。多样性是通过重组A结构域家族表现出的自然变异而形成的。关于进一步的细节,参见Nature Biotechnology 23(12),1556-1561(2005)和Expert Opinion on Investigational Drugs 16(6),909-917(2007年6月)。转铁蛋白是单体血清转运糖蛋白。可以通过在允许的表面环中插入肽序列来工程化改造转铁蛋白,以结合不同的靶抗原。工程化改造的转铁蛋白支架的示例包括反式体。关于进一步的细节,参见J.Biol.Chem 274,24066-24073(1999)。设计的锚蛋白重复序列蛋白(DARPin)来源于锚蛋白,锚蛋白是介导整合膜蛋白与细胞支架的附接的蛋白质家族。单个锚蛋白重复序列是由两个α-螺旋和一个β-转角组成的33残基基序。它们可通过随机化每个重复序列中的第一α-螺旋和β-转角中的残基,而工程化改造成结合不同的靶抗原。它们的结合界面可以通过增加模块的数量来增加(亲和力成熟方法)。关于进一步的细节,参见J.Mol.Biol.332,489-503(2003)、PNAS 100(4),1700-1705(2003)和J.Mol.Biol.369,1015-1028(2007)以及US20040132028A1。单结构域抗体是由单一单体可变抗体结构域组成的抗体片段。第一单结构域来源于骆驼科动物的抗体重链的可变结构域(纳米抗体或VHH片段)。此外,术语单结构域抗体包含自体人重链可变结构域(aVH)或来源于鲨鱼的VNAR片段。纤连蛋白可以经工程化改造以结合抗原的支架。Adnectin由III型人纤连蛋白(FN3)的15个重复单元的第10结构域的天然氨基酸序列的主链组成。β-夹层的一个末端处的三个环可以经工程化改造,以使Adnectin能够特异性识别感兴趣的治疗靶点。关于进一步的细节,参见ProteinEng.Des.Sel.18,435-444(2005)、US20080139791、WO2005056764和US6818418B1。肽适体是组合的识别分子,该组合的识别分子由恒定的支架蛋白,通常是硫氧还蛋白(TrxA)组成,所述恒定的支架蛋白含有在活性位点处插入的受约束的可变肽环。关于进一步的细节,参见Expert Opin.Biol.Ther.5,783-797(2005)。微体来源于含有3至4个半胱氨酸桥、长度为25至50个氨基酸的天然存在的微蛋白,所述微蛋白的示例包括KalataBI和芋螺毒素以及knottin。微蛋白具有环,其可以经工程化改造为包括多达25个氨基酸而不影响微蛋白的整体折叠。关于工程化改造的knottin结构域的进一步细节,参见WO2008098796。
作为参考分子的“结合相同表位的抗原结合分子”是指这样的抗原结合分子,在竞争测定中所述抗原结合分子使参考分子与其抗原的结合被阻断50%或更多,并且相反地,在竞争测定中参考分子使抗原结合分子与其抗原的结合被阻断50%或更多。
术语“抗原结合结构域”是指抗原结合分子的一部分,其包含与抗原的一部分或全部特异性结合并互补的区域。在抗原很大的情况下,抗原结合分子可以仅结合抗原的特定部分,该部分称为表位。抗原结合结构域可以由例如一个或多个可变结构域(也称为可变区)提供。优选地,抗原结合结构域包含抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)。
如本文所用,术语“抗原决定簇”与“抗原”和“表位”同义,并且是指多肽大分子上的位点(例如一段连续的氨基酸或由非连续氨基酸的不同区域组成的构象构型),抗原结合部分与所述位点结合,从而形成抗原结合部分-抗原复合物。有用的抗原决定簇可以在例如肿瘤细胞的表面上、病毒感染细胞的表面上、其他患病细胞的表面上、免疫细胞的表面上、血清中的游离物和/或细胞外基质(ECM)中找到。除非另有说明,否则本文中用作抗原的蛋白质可以是来自任何脊椎动物来源的任何天然形式的蛋白质,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如人类)和啮齿动物(例如小鼠和大鼠)。在一个具体实施例中,抗原是人蛋白质。当提及本文中的特定蛋白质时,该术语涵盖“全长”、未加工的蛋白质,以及由细胞内加工而产生的任何形式的蛋白质。该术语还涵盖天然存在的蛋白质变体,例如剪接变体或等位基因变体。
“特异性结合”是指结合对于抗原具有选择性,并且可以与不需要的或非特异性的相互作用区分开。抗原结合分子与特定抗原结合的能力可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术(例如表面等离子体共振(SPR)技术(在BIAcore仪器上分析)(Liljeblad等人,Glyco J 17,323-329(2000))以及传统的结合测定(Heeley,Endocr Res 28,217-229(2002))来测量。在一个实施例中,例如如通过SPR所测得的,抗原结合分子与不相关蛋白的结合程度小于所述抗原结合分子与抗原的结合程度的约10%。在某些实施例中,与抗原结合的分子的解离常数(Kd)为≤1μM、≤100nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10-8M或更低,例如10-8M至10-13M,例如10-9M至10-13M)。
“亲和力”或“结合亲和力”是指分子(例如抗体)的单个结合位点与其结合配偶体(例如抗原)之间的非共价相互作用的总和的强度。除非另有说明,否则如本文所用的“结合亲和力”是指内在结合亲和力,其反映了结合对的成员(例如抗体和抗原)之间的1:1相互作用。分子X对其配偶体Y的亲和力通常可以用解离常数(Kd)表示,解离常数(Kd)是解离速率常数与缔合速率常数(分别为koff和kon)的比率。因此,等效亲和力可以包括不同的速率常数,只要速率常数的比率保持相同即可。亲和力可以通过本领域已知的常规方法测量,包括本文所述的那些方法。测量亲和力的特定方法是表面等离子体共振(SPR)。
如本文所用的“肿瘤相关抗原”或TAA是指存在于靶细胞表面上的抗原决定簇,该靶细胞为例如肿瘤中的细胞(诸如癌细胞或肿瘤基质的细胞)。在某些方面,靶细胞抗原为肿瘤细胞表面的抗原。在一方面,TAA选自由以下项组成的组:成纤维细胞活化蛋白(FAP)、癌胚抗原(CEA)、叶酸受体α(FolR1)、黑色素瘤相关的硫酸软骨素蛋白多糖(MCSP)、表皮生长因子受体(EGFR)、人表皮生长因子受体2(HER2)和p95HER2。特别地,肿瘤相关抗原为成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)。其他TAA包括HER3、EpCAM、TPBG(5T4)、间皮素、MUC1和PSMA。TAA还包含B细胞表面抗原诸如CD19、CD20和CD79b。此外,也可以包括与多发性骨髓瘤有关的TAA GPRC5D、BCMA和CD38。
术语“成纤维细胞活化蛋白(FAP)”也称为脯氨酰内肽酶FAP或Seprase(EC3.4.21),除非另有说明,否则该术语是指来自任何脊椎动物来源的任何天然FAP,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如人)、非人灵长类动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)。该术语包括“全长”的未加工FAP,以及通过细胞中加工产生的任何形式的FAP。该术语还涵盖FAP的天然存在变体,例如剪接变体或等位基因变体。在一个实施例中,本发明的抗原结合分子能够特异性结合到人、小鼠和/或食蟹猴FAP。人FAP的氨基酸序列示出于UniProt(www.uniprot.org)登录号Q12884(149版,SEQ ID NO:2)或NCBI(www.ncbi.nlm.nih.gov/)RefSeq NP_004451.2中。人FAP的细胞外结构域(ECD)从26位的氨基酸延伸至760位的氨基酸。His标记的人FAP ECD的氨基酸序列显示在SEQ ID NO:135中。小鼠FAP的氨基酸序列示出于UniProt登录号P97321(126版,SEQ ID NO:136)或NCBIRefSeq NP_032012.1中。小鼠FAP的细胞外结构域(ECD)从26位的氨基酸延伸至761位的氨基酸。SEQ ID NO:137示出His标记的小鼠FAP ECD的氨基酸序列。SEQ ID NO:138显示His标记的食蟹猴FAP ECD的氨基酸序列。优选地,本发明的抗FAP结合分子结合到FAP的细胞外结构域。
术语“癌胚抗原(CEA)”也称为癌胚抗原相关细胞粘附分子5(CEACAM5),除非另有说明,否则该术语是指来自任何脊椎动物来源的任何天然CEA,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如人)、非人灵长类动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)。人CEA的氨基酸序列示出于UniProt登录号P06731(151版,SEQ ID NO:3)中。长期以来,CEA被鉴定为一种肿瘤相关的抗原(Gold和Freedman,J Exp Med.,121:439-462,1965;Berinstein N.L.,J Clin Oncol.,20:2197-2207,2002)。CEA最初被归类为仅在胎儿组织中表达的蛋白质,现已在多种正常成人组织中鉴定出来。这些组织主要来源于上皮,包括胃肠道、呼吸道和泌尿生殖道的细胞以及结肠、子宫颈、汗腺和前列腺的细胞(Nap等人,Tumour Biol.,9(2-3):145-53,1988;Nap等人,Cancer Res.,52(8):2329-23339,1992)。上皮来源的肿瘤及其转移均包含CEA作为肿瘤相关抗原。CEA本身的存在并不表示已转化为癌细胞,但CEA的分布具有指示性。在正常组织中,CEA通常在细胞顶面上表达(S.,Semin Cancer Biol.9(2):67-81(1999)),使其无法被血流中的抗体吸收。与正常组织相比,CEA倾向于在癌细胞的整个表面表达(S.,SeminCancer Biol.9(2):67-81(1999))。表达模式的这一变化使CEA易于与癌细胞中的抗体结合。此外,CEA在癌细胞中的表达增加。此外,CEA表达的增加促进细胞间粘附的增加,其可能导致转移(Marshall J.,Semin Oncol.,30(a Suppl.8):30-6,2003)。CEA在各种肿瘤实体中的表达普遍非常高。根据已发表的数据,自己在组织样本中实施的分析证实了CEA的高发性,其中在大肠癌(CRC)中的发生率为约95%,在胰腺癌中的发生率为90%,在胃癌中的发生率为80%,在非小细胞肺癌(NSCLC,与HER3共表达)中的发生率为60%,在乳腺癌中的发生率为40%;并且发现在小细胞肺癌和胶质母细胞瘤中的表达水平低。
CEA易于从细胞表面裂解,并且直接或通过淋巴管从肿瘤中流入血流中。由于这种特性,血清CEA水平已被用作诊断癌症和筛查癌症(尤其是结直肠癌)复发的临床指标(Goldenberg D M.,The International Journal of Biological Markers,7:183-188,1992;Chau I.等人,J Clin Oncol.,22:1420-1429,2004;Flamini等人,Clin Cancer Res;12(23):6985-6988,2006)。
术语“FolR1”是指叶酸受体α,并且已被鉴定为许多癌症的潜在预后和治疗靶点。除非另有说明,否则它是指来自任何脊椎动物来源的任何天然FolR1,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如,人)、非人灵长类动物(例如,食蟹猴)和啮齿动物(例如,小鼠和大鼠)。人FolR1的氨基酸序列显示在UniProt登录号P15328(SEQ ID NO:139)中,鼠FolR1具有UniProt登录号P35846(SEQ ID NO:140)的氨基酸序列,而食蟹猴FolR1具有如UniProt登录号G7PR14(SEQ ID NO:141)中所示的氨基酸序列。FolR1是在细胞的质膜上表达的N-糖基化蛋白。FolR1对叶酸和几种还原的叶酸衍生物具有很高的亲和力,并介导生理叶酸(即,5-甲基四氢叶酸)向细胞内部的传递。FOLR1是FOLR1定向癌症疗法的理想靶点,因为它在绝大多数卵巢癌以及许多子宫癌、子宫内膜癌、胰腺癌、肾癌、肺癌和乳腺癌中均过表达,而FOLR1在正常组织上的表达限定在肾近端小管、肺的肺泡肺细胞、膀胱、睾丸,脉络丛和甲状腺中的上皮细胞的顶膜。最近的研究已经鉴定,FolR1表达在三阴性乳腺癌中特别高(Necela等人PloS One 2015,10(3),e0127133)。
术语“黑素瘤相关硫酸软骨素蛋白多糖(MCSP)”也称为硫酸软骨素蛋白聚糖4(CSPG4),除非另有说明,否则该术语是指来自任何脊椎动物来源的任何天然MCSP,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如人)、非人灵长类动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)。人MCSP的氨基酸序列显示在UniProt登录号Q6UVK1(103版,SEQ IDNO:142)中。MCSP是在细胞膜上表达的高度糖基化的整体膜硫酸软骨素蛋白聚糖,由N-连接的280kDa糖蛋白组分和450-kDa硫酸软骨素蛋白聚糖组分组成(Ross等人,Arch.Biochem.Biophys.1983,225:370-38)。MCSP广泛分布于大量正常细胞和转化细胞中。特别地,在表皮的几乎所有基底细胞中都发现了MCSP。MCSP在黑色素瘤细胞中差异表达,发现其在超过90%的良性痣和黑色素瘤病变中表达。还已经发现MCSP在非黑色素细胞起源的肿瘤(包括基底细胞癌、神经嵴起源的各种肿瘤和乳腺癌)中表达。
术语“表皮生长因子受体(EGFR)”也称为原癌基因c-ErbB-1或受体酪氨酸蛋白激酶erbB-1,除非另有说明,否则该术语是指来自任何脊椎动物来源的任何天然EGFR,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如人)、非人灵长类动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)。人EGFR的氨基酸序列显示在UniProt登录号P00533(211版,SEQ IDNO:143)中。原癌基因“HER2”(人表皮生长因子受体2)编码与人表皮生长因子受体有关并在某种程度上同源的蛋白质酪氨酸激酶(p185HER2)。HER2在本领域也被称为c-erbB-2,有时也被称为大鼠同系物neu。HER2的扩增和/或过表达与多种人类恶性肿瘤相关,并且似乎与25-30%的人类乳腺癌和卵巢癌的进展密切相关。此外,扩增的程度与观察到的患者中位生存时间成反比(Slamon,D.J.等人,Science 244:707-712(1989))。人HER2的氨基酸序列显示在UniProt登录号P04626(230版,SEQ ID NO:144)中。本文所用的术语“p95HER2”是指HER2受体蛋白的羧基末端片段(CTF),也称为“611-CTF”或“100-115kDa p95HER2”。通过在全长HER2分子的密码子位置611处开始HER2 mRNA的翻译,在细胞中生成p95HER2片段(Anido等人,EMBO J 25;3234-44(2006))。它的分子量为100kDa至115kDa,并在细胞膜上表达,在那里它可以形成通过分子间二硫键维持的同型二聚体(Pedersen等人,Mol CellBiol 29,3319-31(2009))。人p95HER2的示例性序列在SEQ ID NO:145中给出。
除非另有说明,否则术语“CD28”(分化簇28,Tp44)是指来自任何脊椎动物来源的任何CD28蛋白,该脊椎动物来源包括哺乳动物诸如灵长类动物(例如,人)、非人灵长类动物(例如,食蟹猴)和啮齿动物(例如,小鼠和大鼠)。CD28在T细胞上表达,并提供T细胞活化和生存所需的共刺激信号。除T细胞受体(TCR)外,还通过CD28刺激T细胞,可以为各种白介素的产生提供有效的信号。CD28是CD80(B7.1)和CD86(B7.2)蛋白的受体,并且是在幼稚T细胞上组成性表达的唯一B7受体。人CD28的氨基酸序列示出于UniProt(www.uniprot.org)登录号P10747(SEQ ID NO:1)中。
“激动性抗体”是指具有针对给定受体的激动功能的抗体。通常,当激动剂配体(因子)与受体结合时,受体蛋白的三级结构改变,并且受体被活化(当受体是膜蛋白时,通常会转导细胞生长信号等)。如果受体是二聚体形成型的受体,那么激动性抗体可以以适当的距离和角度将受体二聚化,从而与配体相似地起作用。适当的抗受体抗体可以模拟通过配体执行的受体二聚化,因此可以成为激动性抗体。
“CD28激动性抗原结合分子”或“CD28常规激动性抗原结合分子”是一种抗原结合分子,在存在T细胞受体信号(“信号2”)的情况下,其模仿CD28天然配体(CD80或CD86)的增强T细胞活化的作用。T细胞需要两个信号才能完全活化。在生理条件下,“信号1”的形成是T细胞受体(TCR)分子与抗原呈递细胞(APC)上的肽/主要组织相容性复合体(MHC)复合物的相互作用,而“信号2”是通过共刺激受体(例如,CD28)的结合而产生的。CD28激动性抗原结合分子能够共刺激T细胞(信号2)。它也能够与对TCR复合物具有特异性的分子联合诱导T细胞增殖和细胞因子分泌,但是CD28激动性抗原结合分子不能在没有额外刺激TCR的情况下完全活化T细胞。然而,存在CD28特异性抗原结合分子的亚类,即所谓的CD28超激动性抗原结合分子。“CD28超激动性抗原结合分子”是能够完全活化T细胞而无需额外刺激TCR的CD28抗原结合分子。通常将超激动剂定义为能够对靶受体产生比内源性激动剂(配体)更大的最大响应的激动剂,因此具有超过100%的效力,但是相对于CD28,CD28超激动性抗原结合分子是指能够在不事先活化T细胞的情况下诱导T细胞增殖和细胞因子分泌的CD28抗原结合分子(信号1)。
术语“可变结构域”或“可变区”是指参与抗原结合分子与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域(分别为VH和VL)通常具有相似的结构,其中每个结构域包含四个保守框架区(FR)和三个高变区(HVR)。参见例如,Kindt等人,Kuby Immunology,第6版,W.H.Freeman and Co.,第91页(2007)。单个VH或VL结构域可足以赋予抗原结合特异性。
如本文所用的术语“高变区”或“HVR”是指抗原结合可变结构域中在序列上高变并确定抗原结合特异性的各个区域,例如“互补决定区”(“CDR”)。通常,抗原结合结构域包含六个CDR:三个在VH中(CDR-H1、CDR-H2、CDR-H3),并且三个在VL中的(CDR-L1、CDR-L2、CDR-L3)。本文中的示例性CDR包括:
(a)在氨基酸残基26-32(L1)、50-52(L2)、91-96(L3)、26-32(H1)、53-55(H2)和96-101(H3)处发生的高可变环(Chothia和Lesk,J.Mol.Biol.196:901-917(1987));
(b)存在于氨基酸残基24-34(L1)、50-56(L2)、89-97(L3)、31-35b(H1)、50-65(H2)和95-102(H3)处的CDR(Kabat等人,Sequences of Proteins of ImmunologicalInterest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991));以及
(c)存在于氨基酸残基27c-36(L1)、46-55(L2)、89-96(L3)、30-35b(H1)、47-58(H2)和93-101(H3)处的抗原接触点(MacCallum等人,J.Mol.Biol.262:732-745(1996))。
除非另有说明,否则CDR根据Kabat等人所述的方法(同上)确定。本领域技术人员将理解,也可以根据Chothia(同上)、McCallum(同上)所述的方法或任何其他在科学上接受的命名法来确定CDR名称。Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以明确地将该“Kabat编号”系统分配给任何可变区序列,而不依赖于序列本身之外的任何实验数据。如本文所用,“Kabat编号”是指由Kabat等人,U.S.Dept.of Health and Human Services,"Sequence of Proteins of ImmunologicalInterest"(1983)所述的编号系统。除非另有说明,否则提及抗体可变区中特定氨基酸残基位置的编号是根据Kabat编号系统。
如本文所用,在抗原结合分子(例如,抗体)的上下文中,术语“亲和力成熟的”是指,(例如,通过突变)衍生自参考抗原结合分子的抗原结合分子与参考抗体结合相同的抗原,优选结合相同的表位;并且对抗原的亲和力高于参考抗原结合分子的亲和力。亲和力成熟通常涉及修饰抗原结合分子的一个或多个CDR中的一个或多个氨基酸残基。通常,亲和力成熟的抗原结合分子与初始参考抗原结合分子结合相同的表位。
“框架”或“FR”是指除高变区(HVR)残基之外的可变结构域残基。可变结构域的FR通常由以下四个FR结构域组成:FR1、FR2、FR3和FR4。因此,HVR和FR序列通常在VH(或VL)中以如下序列出现:FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。
出于本文目的的“受体人框架”是这样的框架,其包含来源于如下所定义的人免疫球蛋白框架或人共有框架的轻链可变结构域(VL)框架或重链可变结构域(VH)框架的氨基酸序列。“来源于”人免疫球蛋白框架或人共有框架的受体人框架可包含与所述人免疫球蛋白框架或人共有框架相同的氨基酸序列,或者其可以包含氨基酸序列变化。在一些实施例中,氨基酸变化的数量为10个或更少、9个或更少、8个或更少、7个或更少、6个或更少、5个或更少、4个或更少、3个或更少,或2个或更少。在一些实施例中,VL受体人框架在序列上与VL人免疫球蛋白框架序列或人共有框架序列相同。
术语“嵌合”抗体是指这样的抗体,在所述抗体中重链和/或轻链的一部分来源于特定来源或物种,而重链和/或轻链的其余部分来源于不同的来源或物种。
抗体的“类别”是指抗体的重链所具有的恒定结构域或恒定区的类型。存在五大类抗体:IgA、IgD、IgE、IgG和IgM,并且这些类别中的若干可以进一步分为亚类(同种型),例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。
“人源化”抗体是指包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在某些实施例中,人源化抗体将基本上包含所有中的至少一个可变结构域,通常是两个可变结构域,其中所有或基本上所有HVR(例如CDR)对应于非人抗体的HVR,并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。“人源化形式”的抗体,例如非人抗体,是指已经经历进行过人源化的抗体。本发明涵盖的其他形式的“人源化抗体”是这样的抗体,相对于原始抗体,所述抗体中的恒定区已经经过了另外修饰或改变,以产生根据本发明的特性,特别是关于C1q结合和/或Fc受体(FcR)结合的特性。
“人”抗体是这样的抗体,其具有的氨基酸序列与由人或人细胞产生的或来源于利用人抗体库或其他人抗体编码序列的非人来源的抗体的氨基酸序列相对应。人抗体的该定义特别地排除了包含非人抗原结合残基的人源化抗体。
本文中的术语“Fc结构域”或“Fc区”用于定义含有恒定区的至少一部分的抗体重链C末端区域。该术语包括天然序列Fc区和变体Fc区。IgG Fc区包含IgG CH2结构域和IgGCH3结构域。人IgG Fc区的“CH2结构域”通常从大致231位的氨基酸残基延伸至大致340位的氨基酸残基。在一个实施例中,碳水化合物链附接至CH2结构域。本文的CH2结构域可以是天然序列CH2结构域或变体CH2结构域。“CH3结构域”包含Fc区中CH2结构域C末端的一段残基(即,从IgG的约341位的氨基酸残基到约447位的氨基酸残基)。本文的CH3区可以是天然序列CH3结构域或变体CH3结构域(例如在一条链中具有引入的“凸起”(“突起”)而在另一条链中具有相应的引入的“空腔”(“孔”)的CH3结构域;参见以引用方式明确并入本文的美国专利号5,821,333)。此类变体CH3结构域可用于促进如本文所述的两条不相同的抗体重链的异二聚化。在一个实施例中,人IgG重链Fc区从Cys226或从Pro230延伸至重链的羧基末端。然而,Fc区的C末端赖氨酸(Lys447)可以存在或不存在。除非本文另外规定,否则Fc区或恒定区中氨基酸残基的编号是根据EU编号系统,EU编号系统也称为EU索引,如在Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述。
“杵臼结构(knob-into-hole)”技术描述于例如US 5,731,168;US 7,695,936;Ridgway等人,Prot Eng 9,617-621(1996)和Carter,J Immunol Meth 248,7-15(2001)中。通常,该方法涉及在第一多肽的界面处引入凸起(“突起”)并在第二多肽的界面中引入相应的空腔(“孔”),使得所述凸起可以定位在所述空腔中,以便促进异二聚体的形成并阻碍同二聚体的形成。凸起是通过用较大侧链(例如酪氨酸或色氨酸)取代来自第一多肽的界面的小氨基酸侧链而构建的。具有与凸起相同或相似大小的补偿空腔是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在第二多肽的界面中创建的。凸起和空腔可以通过改变编码多肽的核酸来制备,例如通过位点特异性诱变或通过肽合成。在一个具体实施例中,突起修饰包含Fc结构域的两个亚基中的一个中的氨基酸取代T366W,而孔修饰包含Fc结构域的两个亚基中的另一个中的氨基酸取代T366S、L368A和Y407V。在另一个具体实施例中,包含突起修饰的Fc结构域的亚基另外包含氨基酸取代S354C,而包含孔修饰的Fc结构域的亚基另外包含氨基酸取代Y349C。引入这两个半胱氨酸残基导致在Fc区的两个亚基之间形成二硫桥,从而进一步稳定化二聚体(Carter,J Immunol Methods 248,7-15(2001))。
“与免疫球蛋白的Fc区等同的区域”旨在包括免疫球蛋白的Fc区的天然存在的等位基因变体,以及具有产生取代、添加或缺失但基本上不降低免疫球蛋白介导效应子功能(诸如抗体依赖性细胞毒性)的能力的修改的变体。例如,可以使一个或多个氨基酸从免疫球蛋白的Fc区的N-末端或C-末端缺失,而基本上不丧失生物学功能。可以根据本领域中已知的一般规则来选择此类变体,以便对活性具有最小的影响(参见例如Bowie,J.U.等人,Science 247:1306-10(1990))。
术语“效应子功能”是指可归因于抗体的Fc区、随着抗体同种型的变化而变化的那些生物活性。抗体效应子功能的示例包括:C1q结合和补体依赖性细胞毒性(CDC)、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞的抗原摄取、下调细胞表面受体(例如B细胞受体),以及B细胞活化。
Fc受体结合依赖性效应子功能可通过抗体的Fc区与Fc受体(FcR)的相互作用来介导,Fc受体是造血细胞上的特异性细胞表面受体。Fc受体属于免疫球蛋白超家族,并且已被证明可通过吞噬免疫复合物来介导去除抗体包被的病原体,并且通过抗体依赖性细胞介导的细胞毒性(ADCC)来裂解涂有相应抗体的红细胞及其他各种细胞靶点(例如,肿瘤细胞)(参见例如Van de Winkel,J.G.和Anderson,C.L.,J.Leukoc.Biol.49(1991)511-524)。FcRs由其对免疫球蛋白同种型的特异性来限定:IgG抗体的Fc受体称为FcγR。Fc受体结合描述于例如:Ravetch,J.V.和Kinet,J.P.,Annu.修订版,Immunol.9(1991)457-492;Capel,P.J.等人,Immunomethods 4(1994)25-34;de Haas,M.等人,J.Lab.Clin.Med.126(1995)330-341;以及Gessner,J.E.等人,Ann.Hematol.76(1998)231-248。
IgG抗体(FcγR)Fc区受体的交联触发多种效应子功能,包括吞噬作用、抗体依赖性细胞细胞毒性、炎症介质的释放以及免疫复合物清除和抗体产生的调节。已在人体中鉴定出三类FcγR,其中包括:
-FcγRI(CD64)以高亲和力结合单体IgG,并且在巨噬细胞、单核细胞、中性粒细胞和嗜酸性粒细胞上表达。Fc区IgG中在至少一个氨基酸残基E233-G236、P238、D265、N297、A327和P329(根据Kabat的EU索引编号)的修饰,降低与FcγRI的结合。在233-236位的IgG2残基被IgG1和IgG4取代,使得与FcγRI的结合力降低103倍,并且消除了人单核细胞对抗体敏化红细胞的应答(Armour,K.L.等人,Eur.J.Immunol.29(1999)2613–2624)。
-FcγRII(CD32)以中等至低亲和力结合复合IgG,并得到广泛表达。该受体可分为两种亚型,即FcγRIIA和FcγRIIB。FcγRIIA存在于许多参与杀死的细胞(例如,巨噬细胞、单核细胞、中性粒细胞)中,并且似乎能够活化杀死过程。FcγRIIB似乎在抑制过程中起作用,并且存在于B细胞、巨噬细胞以及肥大细胞和嗜酸性粒细胞中。在B细胞上,它似乎起到抑制免疫球蛋白进一步产生以及同种型转换为例如IgE类的作用。在巨噬细胞上,FcγRIIB用于抑制由FcγRIIA介导的吞噬作用。在嗜酸性粒细胞和肥大细胞上,B型可能通过IgE与其单独受体的结合而有助于抑制这些细胞的活化。发现例如抗体(包含在至少一个氨基酸残基E233-G236、P238、D265、N297、A327、P329、D270、Q295、A327、R292和K414(根据Kabat的EU索引编号)的突变的IgG Fc区)对FcγRIIA的结合力降低。
-FcγRIII(CD16)以中等至低亲和力结合IgG,并且包括两种类型。FcγRIIIA存在于NK细胞、巨噬细胞、嗜酸性粒细胞以及一些单核细胞和T细胞上,并且介导ADCC。FcγRIIIB在中性粒细胞上的表达水平高。发现例如抗体(包含具有至少在氨基酸残基E233-G236、P238、D265、N297、A327、P329、D270、Q295、A327、S239、E269、E293、Y296、V303、A327、K338和D376(根据Kabat的EU索引编号)中的一个的突变的IgG Fc区)对FcγRIIIA的结合力降低。
Shields,R.L.等人(J.Biol.Chem.276(2001)6591-6604)描述了人IgG1上与Fc受体的结合位点的定位、上述突变位点以及测量与FcγRI和FcγRIIA结合的方法。
术语“ADCC”或“抗体依赖性细胞毒性”是导致免疫效应细胞裂解抗体包被的靶细胞的免疫机制。靶细胞是与包含Fc区的抗体或其衍生物特异性结合的细胞,该特异性结合通常是通过Fc区的N末端的蛋白质部分。如本文所用,术语“降低的ADCC”被定义为在靶细胞周围的培养基中给定抗体浓度下在给定时间内通过上面定义的ADCC机制裂解的靶细胞数量减少,和/或在靶细胞周围的培养基中通过ADCC机制在给定时间内实现对给定数量的靶细胞的裂解所必需的抗体浓度增加。ADCC降低是相对于使用相同的标准生产、纯化、配制和储存方法(该等方法为本领域技术人员已知的),由相同类型的宿主细胞产生但尚未被工程化的相同抗体介导的ADCC。例如,由在其Fc结构域中包含降低ADCC的氨基酸取代的抗体介导的ADCC的降低是相对于由在Fc结构域中没有该氨基酸取代的相同抗体介导的ADCC。用于测量ADCC的合适测定法是本领域中熟知的(参见例如PCT公开号WO 2006/082515或PCT公开号WO 2012/130831)。例如,通过测量抗体与表达Fcγ受体的细胞(诸如重组表达FcγRI和/或FcγRIIA或NK细胞(在本质上表达FcγRIIIA)的细胞)的结合来研究抗体诱导介导ADCC的初始步骤的能力。具体地,测量与NK细胞上与FcγR的结合。
“活化Fc受体”是这样的Fc受体,其在抗体的Fc区接合后,引起刺激携带受体的细胞执行效应子功能的信号传导事件。活化Fc受体包括FcγRIIIa(CD16a)、FcγRI(CD64)、FcγRIIa(CD32)和FcαRI(CD89)。特定的活化Fc受体是人FcγRIIIa(参见UniProt登录号P08637,版本141)。
“胞外域”是延伸到细胞外空间(即靶细胞外部的空间)的膜蛋白的结构域。胞外域通常是蛋白质的部分,其开始与表面的接触,而该接触导致信号转导。
术语“肽连接基”是指包含一个或多个氨基酸,通常约2至20个氨基酸的肽。肽连接基是本领域中已知的或在本文中描述的。合适的非免疫原性连接基肽是例如(G4S)n、(SG4)n或G4(SG4)n肽连接基,其中“n”通常为介于1与5之间、通常介于2与4之间的数字,特别地是2,即选自由以下项组成的组的肽:GGGGS(SEQ ID NO:146)、GGGGSGGGGS(SEQ ID NO:147)、SGGGGSGGGG(SEQ ID NO:148)和GGGGSGGGGSGGGG(SEQ ID NO:149),但也包括以下序列:GSPGSSSSGS(SEQ ID NO:150)、(G4S)3(SEQ ID NO:151)、(G4S)4(SEQ ID NO:152)、GSGSGSGS(SEQ ID NO:153)、GSGSGNGS(SEQ ID NO:154)、GGSGSGSG(SEQ ID NO:155)、GGSGSG(SEQ IDNO:156)、GGSG(SEQ ID NO:157)、GGSGNGSG(SEQ ID NO:158)、GGNGSGSG(SEQ ID NO:159)和GGNGSG(SEQ ID NO:160)。特定目标肽连接基为(G4S)(SEQ ID NO:146)、(G4S)2或GGGGSGGGGS(SEQ ID NO:147)、(G4S)3(SEQ ID NO:151)和(G4S)4(SEQ ID NO:152)。
如本申请中所用的术语“氨基酸”表示包括以下项的天然存在的羧基α-氨基酸的组:丙氨酸(三字母代码:ala,单字母代码:A)、精氨酸(arg,R)、天冬酰胺(asn,N)、天冬氨酸(asp,D)、半胱氨酸(cys,C)、谷氨酰胺(gln,Q)、谷氨酸(glu,E)、甘氨酸(gly,G)、组氨酸(his,H)、异亮氨酸(ile,I)、亮氨酸(leu,L)、赖氨酸(lys,K)、蛋氨酸(met,M)、苯丙氨酸(phe,F)、脯氨酸(pro,P)、丝氨酸(ser,S)、苏氨酸(thr,T)、色氨酸(trp,W)、酪氨酸(tyr,Y),以及缬氨酸(val,V)。
“融合”或“联结”意指组分(例如,多肽和所述TNF配体家族成员的胞外域)直接地或经由一个或多个肽连接基而通过肽键连接。
相对于参考多肽(蛋白质)序列的“氨基酸序列一致性百分比(%)”被定义为在比对候选序列中的氨基酸残基与参考多肽序列中的氨基酸残基并引入缺口(如果需要的话)以实现最大的序列一致性百分比后,并且在不将任何保守取代考虑为所述序列一致性的一部分的情况下,与所述参考多肽序列中的氨基酸残基相同的所述候选序列中的氨基酸残基的百分比。用于确定氨基酸序列一致性百分比的比对可以以本领域技术范围内的各种方式实现,例如使用公众可获得的计算机软件,诸如BLAST、BLAST-2、ALIGN.SAWI或Megalign(DNASTAR)软件。本领域技术人员可确定用于比对序列的适当参数,包括在所比较的序列的全长上实现最大比对所需的任何算法。然而,为了本文的目的,使用序列比较计算机程序ALIGN-2来生成氨基酸序列一致性%的值。ALIGN-2序列比较计算机程序由Genentech,Inc.编写,并且源代码已经与用户文档一起提交到U.S.Copyright Office,Washington D.C.,20559,在那里以美国版权登记号TXU510087注册。ALIGN-2程序可从Genentech,Inc.,SouthSan Francisco,California公开获得,或者可以从所述源代码编译。ALIGN-2程序应经编译以在UNIX操作系统上使用,所述UNIX操作系统包括数字UNIX V4.0D。所有序列比较参数均由ALIGN-2程序设置并且不变。在采用ALIGN-2进行氨基酸序列比较的情况下,给定氨基酸序列A与给定氨基酸序列B的氨基酸序列一致性%(其可以替代地表达为给定氨基酸序列A具有或包含与给定氨基酸序列B的某一氨基酸序列一致性%)计算如下:
100乘以分数X/Y
其中X是由序列比对程序ALIGN-2在该程序对A和B的比对中评分为相同匹配的氨基酸残基的数目,而其中Y是B中氨基酸残基的总数。应当理解,在氨基酸序列A的长度不等于氨基酸序列B的长度的情况下,A与B的氨基酸序列一致性%将不等于B与A的氨基酸序列一致性%。除非另外特别指明,否则本文所使用的所有氨基酸序列一致性%的值是如前一段中所述使用ALIGN-2计算机程序获得的。
在某些实施例中,考虑了本文提供的CD28抗原结合分子的氨基酸序列变体。例如,可能需要改善CD28抗原结合分子的结合亲和力和/或其他生物学特性。CD28抗原结合分子的氨基酸序列变体可以通过向编码分子的核苷酸序列中引入适当的修饰或通过肽合成来制备。此类修饰包括例如抗体氨基酸序列内残基的缺失、和/或插入和/或取代。可以进行缺失、插入和取代的任何组合以实现最终构建体,前提条件是最终构建体具有所需特征,例如抗原结合。用于取代诱变的感兴趣的位点包括HVR和框架(FR)。保守性取代在表B中表头“优选的取代”下提供,并在下文中参考氨基酸侧链类别(1)至(6)进一步描述。可以将氨基酸取代引入感兴趣的分子中,并对产物进行所需活性(例如保留/改善的抗原结合、降低的免疫原性,或改善的ADCC或CDC)筛选。
表A
可根据共同的侧链特性将氨基酸分组:
(1)疏水性:正亮氨酸、Met、Ala、Val、Leu、Ile;
(2)中性亲水性:Cys、Ser、Thr、Asn、Gln;
(3)酸性:Asp、Glu;
(4)碱性:His、Lys、Arg;
(5)影响链取向的残基:Gly、Pro;
(6)芳香族:Trp、Tyr、Phe。
非保守性取代将需要用这些类别中的一个的成员交换另一类别。
术语“氨基酸序列变体”包括实质性变体,其中在亲本抗原结合分子(例如人源化或人抗体)的一个或多个高变区残基中存在氨基酸取代。通常,相对于亲本抗原结合分子,选为用于进一步研究的一个或多个所得变体将在某些生物学特性方面(例如,亲和力增加、免疫原性降低)有改变(例如,改善)和/或将基本上保留亲本抗原结合分子的某些生物学特性。示例性取代变体是亲和力成熟抗体,其可例如使用诸如本文所述的那些基于噬菌体展示的亲和力成熟技术方便地生成。简而言之,将一个或多个HVR残基突变并且将变体抗原结合分子展示在噬菌体上并针对特定生物活性(例如结合亲和力)进行筛选。在某些实施例中,取代、插入或缺失可发生在一个或多个HVR内,只要此类改变基本上不降低抗原结合分子的抗原结合能力即可。例如,可在HVR中进行基本上不降低结合亲和力的保守性改变(例如,如本文提供的保守性取代)。可用于鉴别可被靶向诱变的抗体残基或区域的方法称作“丙氨酸扫描诱变”,如Cunningham和Wells(1989)Science,244:1081-1085所述。在此方法中,鉴别残基或一组靶残基(例如,带电残基,诸如Arg、Asp、His、Lys和Glu)并用中性或带负电的氨基酸(例如,丙氨酸或多丙氨酸)替换以确定抗体与抗原的相互作用是否受到影响。可在对初始取代展现功能敏感性的氨基酸位置引入其他取代。另选地或另外地,利用抗原-抗原结合分子复合物的晶体结构鉴别抗体与抗原之间的接触点。可靶向或消除作为取代的候选的此类接触残基和相邻残基。可筛选变体以确定它们是否具备期望的特性。
氨基酸序列插入包括长度范围为一个残基至含有一百个或更多个残基的多肽的氨基和/或羧基末端融合,以及一个或多个氨基酸残基的序列内插入。插入的实例包括具有与增加CD28抗原结合分子的血清半衰期的多肽的N末端或C末端的融合的CD28抗原结合分子。
在某些实施例中,改变本文提供的CD28抗原结合分子以增加或降低抗体糖基化的程度。可通过改变氨基酸序列,使得产生或去除一个或多个糖基化位点,从而便利地获得分子的糖基化变体。当激动性ICOS结合分子包含Fc结构域时,附接于其上的碳水化合物可以被改变。由哺乳动物细胞产生的天然抗体通常包含具有支链的双触角寡糖,所述双触角寡糖通常通过N-连接附接于Fc区的CH2结构域的Asn297。参见,例如,Wright等人TIBTECH 15:26-32(1997)。寡糖可包括各种碳水化合物,例如,甘露糖、N-乙酰基葡糖胺(GlcNAc)、半乳糖和唾液酸,以及附接至双触角寡糖结构的“主干”中的GlcNAc的岩藻糖。在一些实施例中,可以对激动性ICOS结合分子中的寡糖进行修饰,以产生具有某些改善的特性的变体。在一个方面,提供了激动性ICOS结合分子的变体,其具有缺乏附接(直接或间接)至Fc区的岩藻糖的碳水化合物结构。此类岩藻糖基化变体可具有改善的ADCC功能,参见例如美国专利公开号US 2003/0157108(Presta,L.)或US 2004/0093621(协和发酵工业株式会社(KyowaHakko Kogyo Co.,Ltd))。本发明的CD28抗原结合分子的其他变体包括具有两分型寡糖的变体,例如其中附接至Fc区的双触角寡糖是通过GlcNAc两分的。此类变体可具有降低的岩藻糖基化和/或改善的ADCC功能,参见例如WO 2003/011878(Jean-Mairet等人);美国专利号6,602,684(Umana等人);以及US 2005/0123546(Umana等人)。还提供了在附接至Fc区的寡糖中具有至少一个半乳糖残基的变体。此类抗体变体可具有改善的CDC功能并描述于例如WO 1997/30087(Patel等人);WO 1998/58964(Raju,S.);以及WO 1999/22764(Raju,S.)中。
在某些实施例中,可期望产生本发明的CD28抗原结合分子的半胱氨酸工程化改造的变体,例如“thioMAb”,其中分子的一个或多个残基被半胱氨酸残基取代。在特定实施例中,取代的残基存在于分子的可接近位点。通过用半胱氨酸取代那些残基,反应性硫醇基团由此定位于抗体的可接近位点,并且可用于将抗体与其他部分,诸如药物部分或连接基-药物部分缀合,以产生免疫缀合物。在某些实施例中,可用半胱氨酸取代下列残基中的任何一个或多个:轻链的V205(Kabat编号);重链的A118(EU编号);以及重链Fc区的S400(EU编号)。可如例如美国专利号7,521,541中所述形成半胱氨酸工程化改造的抗原结合分子。
在某些方面,可进一步修饰本文提供的CD28抗原结合分子以使其含有本领域已知且易于获得的另外的非蛋白质部分。适合于抗体衍生化的部分包括但不限于水溶性聚合物。水溶性聚合物的非限制性示例包括但不限于聚乙二醇(PEG)、乙二醇/丙二醇的共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二氧戊环、聚-1,3,6-三噁烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或随机共聚物)和葡聚糖或聚(n-乙烯吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/环氧乙烷共聚物、聚氧乙烯化多元醇(例如甘油)、聚乙烯醇以及它们的混合物。由于其在水中的稳定性,聚乙二醇丙醛在制造中可具有优势。聚合物可具有任何分子量,并且可以具有支链或不具有支链。附接至抗体的聚合物的数目可变化,并且如果附接了多于一个聚合物,那么它们可以是相同或不同的分子。通常,可基于以下考虑因素确定用于衍生化的聚合物的数目和/或类型,包括但不限于抗体待改善的特定特性或功能、双特异性抗体衍生物是否将用于限定条件下的疗法等。在另一方面,提供了可通过暴露于辐射而被选择性加热的抗体和非蛋白质性部分的缀合物。在一个实施例中,非蛋白质性部分是碳纳米管(Kam,N.W.等人,Proc.Natl.Acad.Sci.USA 102(2005)11600-11605)。辐射可具有任何波长,并且包括但不限于对普通细胞没有伤害,但是将非蛋白质性部分加热至抗体-非蛋白质性部分附近的细胞被杀死的温度的波长。在另一方面,可以获得本文提供的CD28抗原结合分子的免疫缀合物。“免疫缀合物”是与一种或多种异源分子,包括但不限于细胞毒性剂缀合的抗体。
术语“多核苷酸”是指分离的核酸分子或构建体,例如信使RNA(mRNA)、病毒来源的RNA或质粒DNA(pDNA)。多核苷酸可包含常规磷酸二酯键或非常规键(例如酰胺键,诸如在肽核酸(PNA)中存在的)。术语“核酸分子”是指存在于多核苷酸中的任何一个或多个核酸区段,例如DNA或RNA片段。
关于“分离的”核酸分子或多核苷酸,是指已经从其天然环境中移除的核酸分子,DNA或RNA。例如,编码包含在载体中的多肽的重组多核苷酸出于本发明的目的被视为分离的。分离的多核苷酸的另外的实施例包括维持在异源宿主细胞中的重组多核苷酸或处于溶液中的纯化的(部分地或基本上纯化的)多核苷酸。分离的多核苷酸包括多核苷酸分子,该多核苷酸分子包含在通常包含多核苷酸分子的细胞中,但该多核苷酸分子存在于染色体外或存在于与其天然染色体位置不同的染色体位置上。分离的RNA分子包括本发明的体内或体外RNA转录物,以及正链和负链形式和双链形式。根据本发明的分离的多核苷酸或核酸还包括通过合成产生的此类分子。此外,多核苷酸或核酸可以是或可包括调控元件,诸如启动子、核糖体结合位点或转录终止子。
关于与本发明的参考核苷酸序列具有至少例如95%“一致”的核苷酸序列的核酸或多核苷酸,是指除了多核苷酸序列可包括参考核苷酸序列的每100个核苷酸至多五个点突变之外,多核苷酸的核苷酸序列与参考序列是一致的。换句话讲,为了获得具有与参考核苷酸序列至少95%一致的核苷酸序列的多核苷酸,参考序列中至多5%的核苷酸可缺失或被另外的核苷酸取代,或参考序列中总核苷酸的至多5%的数量的核苷酸可插入到参考序列中。参考序列的这些改变可发生在参考核苷酸序列的5’或3’末端位置或那些末端位置之间的任意位置,或单个地散布在参考序列的残基之中,或以一个或多个连续的组散布在参考序列内。作为一种实际情况,可以使用已知的计算机程序,诸如上文针对多肽所讨论的程序(例如ALIGN-2),常规确定任何特定多核苷酸序列是否与本发明的核苷酸序列至少80%、85%、90%、95%、96%、97%、98%或99%一致。
术语“表达盒”是指通过重组或合成生成的多核苷酸,其具有允许特定核酸在靶细胞中转录的一系列特定核酸元件。重组表达盒可以掺入质粒、染色体、线粒体DNA、质粒DNA、病毒或核酸片段中。典型地,表达载体的重组表达盒部分除其他序列之外还包括待转录的核酸序列和启动子。在某些实施例中,本发明的表达盒包含编码本发明的双特异性抗原结合分子或其片段的多核苷酸序列。
术语“载体”或“表达载体”与“表达构建体”是同义的并且是指用于将特定基因引入与其可操作地关联的靶细胞中并指导所述基因的表达的DNA分子。该术语包括作为自我复制核酸结构的载体,以及整合入其已被引入的宿主细胞的基因组中的载体。本发明的表达载体包含表达盒。表达载体允许大量稳定mRNA的转录。一旦表达载体处于靶细胞内部,即通过细胞转录和/或翻译机制产生由该基因编码的核糖核酸分子或蛋白。在一个实施例中,本发明的表达载体包含表达盒,所述表达盒包含编码本发明的双特异性抗原结合分子或其片段的多核苷酸序列。
术语“宿主细胞”“宿主细胞系”和“宿主细胞培养物”可互换使用,并且是指外源核酸已被引入其中的细胞,包括此类细胞的子代。宿主细胞包括“转化体”和“转化细胞”,其包括原代转化细胞和来源于所述原代转化细胞的子代,不考虑传代次数。子代可能不与亲本细胞的核酸内容物完全一致,而是可能含有突变。本文包括如在原始转化细胞中筛选或选择的具有相同功能或生物活性的突变子代。宿主细胞是可以用于生成本发明的双特异性抗原结合分子的任何类型的细胞系统。宿主细胞包括培养的细胞,举几个例子,例如培养的哺乳动物细胞,诸如CHO细胞、BHK细胞、NS0细胞、SP2/0细胞、YO骨髓瘤细胞、P3X63小鼠骨髓瘤细胞、PER细胞、PER.C6细胞或杂交瘤细胞、酵母细胞、昆虫细胞和植物细胞,以及包括在转基因动物、转基因植物或培养的植物或动物组织中的细胞。
药剂的“有效量”是指在其所施用的细胞或组织中产生生理学变化所需的量。
药剂(例如药物组合物)的“治疗有效量”是指在必需的剂量和时段上有效实现期望的治疗或预防结果的量。治疗有效量的药剂例如消除、减少、延迟、最小化或预防疾病的不良影响。
“个体”或“受试者”是哺乳动物。哺乳动物包括但不限于驯养的动物(例如牛、绵羊、猫、犬和马)、灵长类动物(例如人和非人灵长类动物,诸如猴)、兔以及啮齿类动物(例如小鼠和大鼠)。具体地,个体或受试者是人。
术语“药物组合物”是指处于允许包含在其中的活性成分的生物活性有效的形式,并且不含对于将被施用配制剂的受试者具有不可接受的毒性的另外组分的制剂。
“药用赋形剂”是指药物组合物中除有效成分之外的成分,其对受试者无毒。药用赋形剂包括但不限于缓冲剂、稳定剂或防腐剂。
术语“包装插页”用于指治疗产品的商业包装中通常包括的说明书,其含有涉及此类治疗产品的使用的有关适应症、用法、剂量、施用、联合疗法、禁忌症和/或警告的信息。
如本文所用,“治疗(treatment)”(及其语法变型,诸如“治疗(treat)”或“治疗(treating)”)是指试图改变所治疗个体的自然进程的临床干预,并且可以是为了预防或在临床病理学的进程中进行。治疗的期望效果包括但不限于预防疾病的发生或复发、减轻症状、削弱疾病的任何直接或间接病理学后果、预防转移、降低疾病进展的速率、改善或减轻疾病状态,以及缓解或改善预后。在一些实施例中,本发明的分子用于延迟疾病的发展或用于减缓疾病的进展。
术语“癌症”是指或描述哺乳动物中通常以细胞生长/增殖不受控制为特征的生理状况。因此,本文所用的术语癌症是指增生性疾病,诸如癌、淋巴瘤(例如,霍奇金氏淋巴瘤和非霍奇金氏淋巴瘤)、母细胞瘤、肉瘤和白血病。特别地,术语癌症包括淋巴细胞性白血病、肺癌、非小细胞肺(NSCL)癌、支气管肺泡细胞肺癌、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内黑色素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌(stomach cancer)、胃癌(gastric cancer)、结肠癌、乳腺癌、子宫癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金氏病、食管癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、前列腺癌、膀胱癌、肾癌或输尿管癌、肾细胞癌、肾盂癌、间皮瘤、肝细胞癌、胆管癌、中枢神经系统(CNS)肿瘤、脊椎轴肿瘤、脑干胶质瘤、多形性成胶质细胞瘤、星形细胞瘤、神经鞘瘤、室管膜瘤、成髓细胞瘤、脑膜瘤、鳞状细胞癌、垂体腺瘤和尤文氏肉瘤,包括以上癌症中的任一种的难治性型式,或一种或多种以上癌症的组合。在一方面,癌症是实体瘤。在另一方面,癌症是血液学癌症,特别是白血病,最特别是急性淋巴细胞性白血病(ALL)或急性骨髓性白血病(AML)。
应理解,本发明的所有方面和实施例描述的术语“包含”可以替换为“由以下组成”及“基本上由以下组成”所指的方面和实施例。
本发明的超激动性CD28抗原结合分子
本发明提供了新型超激动性CD28抗原结合分子,其具有特别有利的性质,诸如可生产性、稳定性、结合亲和力、生物活性、靶向效率、降低的毒性、可以给予患者的扩展剂量范围以及从而可能增强的功效。新型超激动性CD28抗原结合分子包含由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个氨基酸取代,该氨基酸取代降低抗原结合分子对Fc受体的结合亲和力和/或效应子功能(Fc沉默),因此避免了经由Fc受体的非特异性交联。相反,它们包含至少一种能够与肿瘤相关抗原诸如成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)特异性结合的抗原结合结构域,该抗原结合结构域在肿瘤部位引起交联。因此,实现了肿瘤特异性T细胞活化。
本文提供了超激动性CD28抗原结合分子,其能够与CD28多价结合并且包含
(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,
(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,和
(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代。
在一个特定方面,超激动性CD28抗原结合分子能够与CD28二价结合并且包含能够与CD28特异性结合的两个抗原结合结构域。
在一方面,提供了前文定义的超激动性CD28抗原结合分子,其中Fc结构域是IgG,特别是IgG1 Fc结构域或IgG4 Fc结构域。在一个特定方面,由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域是IgG1 Fc结构域。Fc结构域包含降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的一个或多个氨基酸取代。在一方面,Fc结构域包含氨基酸取代L234A和L235A(根据Kabat的EU索引编号)。在一方面,Fc结构域属于人IgG1亚类并且包含氨基酸突变L234A、L235A和P329G(根据Kabat EU索引编号)。
在一方面,提供了如前文所定义的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(i)重链可变区(VHCD28),其包含SEQ ID NO:20的重链互补决定区CDR-H1、SEQ IDNO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的轻链互补决定区CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3;或
(ii)重链可变区(VHCD28),其包含SEQ ID NO:36的CDR-H1、SEQ ID NO:37的CDR-H2和SEQ ID NO:38的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:39的CDR-L1、SEQ ID NO:40的CDR-L2和SEQ ID NO:41的CDR-L3。
在一方面,超激动性CD28抗原结合分子的能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:36的CDR-H1、SEQ ID NO:37的CDR-H2和SEQ ID NO:38的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:39的CDR-L1、SEQ ID NO:40的CDR-L2和SEQ ID NO:41的CDR-L3。
在另一方面,超激动性CD28抗原结合分子的能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:20的CDR-H1、SEQ ID NO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3。
此外,提供了如前文所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含与SEQ ID NO:26的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCD28),其包含与SEQ ID NO:27的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
在进一步方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含选自由SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51组成的组的氨基酸序列;以及轻链可变区(VLCD28),其包含选自由SEQ ID NO:27、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61组成的组的氨基酸序列。
在另一方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(a)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(b)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(c)重链可变区(VHCD28),其包含SEQ ID NO:51的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:61的氨基酸序列,或
(d)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(e)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(f)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(g)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(h)重链可变区(VHCD28),其包含SEQ ID NO:43的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(i)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(j)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(k)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列。
在一个特定方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列;以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列。
在另一个特定方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列;以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列。
在进一步特定方面,提供了超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列;以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列。
在进一步方面,提供了如前文所定义的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均为Fab片段。
在一方面,提供了超激动性CD28抗原结合分子,其中能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与癌胚抗原(CEA)特异性结合的抗原结合结构域。
在一方面,提供了如本文所述的超激动性CD28抗原结合分子,其中能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含:(i)CDR-H1,其包含SEQ IDNO:127的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:128的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:129的氨基酸序列;以及轻链可变区(VLCEA),其包含:(iv)CDR-L1,其包含SEQ ID NO:130的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:131的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:132的氨基酸序列。特别地,能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含与SEQ ID NO:133的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCEA),其包含与SEQ IDNO:134的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
在另一方面,提供了超激动性CD28抗原结合分子,其中能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与成纤维细胞活化蛋白(FAP)特异性结合的抗原结合结构域。
在一方面,提供了如本文所述的超激动性CD28抗原结合分子,其中能够与FAP特异性结合的抗原结合结构域包含
(a)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:12的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:13的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:14的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:15的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:16的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:17的氨基酸序列;或
(b)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:4的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:5的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:6的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:7的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:8的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:9的氨基酸序列。特别地,能够与FAP特异性结合的抗原结合结构域包含:重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:12的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:13的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:14的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:15的氨基酸序列,(v)CDR-L2,其包含SEQID NO:16的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:17的氨基酸序列。
在一方面,提供了超激动性CD28抗原结合分子,其中能够与FAP特异性结合的抗原结合结构域包含:(a)重链可变区(VHFAP),其包含与SEQ ID NO:18的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ ID NO:19的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;或者(b)重链可变区(VHFAP),其包含与SEQ ID NO:10的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ ID NO:11的氨基酸序列具有至少约95%、96%、97%、98%、99%或100%同一性的氨基酸序列。特别地,能够与FAP特异性结合的抗原结合结构域包含:重链可变区(VHFAP),其包含SEQ ID NO:18的氨基酸序列;以及轻链可变区(VLFAP),其包含SEQ ID NO:19的氨基酸序列。
对于与CD28结合为二价并且对于与肿瘤相关抗原结合为单价(1+2格式)的超激动性CD28抗原结合分子
在另一方面,提供了如本文所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的VH和VL结构域,其中所述VH结构域经由肽连接基与两条重链中的一条的C末端联结,并且其中所述VL结构域经由肽连接基与第二重链的C末端联结。
在一方面,肽连接基包含选自SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:151和SEQ ID NO:152的氨基酸序列。更特别地,肽连接基包含SEQ ID NO:152。
在另一方面,超激动性CD28抗原结合分子包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的VH和VL结构域,其中VH结构域经由肽连接基与Fc杵重链的C末端联结,并且其中VL结构域经由肽连接基与Fc臼重链的C末端联结。
在一个特定方面,提供了超激动性CD28抗原结合分子,其包含:两条各自包含SEQID NO:62的氨基酸序列的轻链、包含SEQ ID NO:71的氨基酸序列的第一重链和包含SEQ IDNO:72的氨基酸序列的第二重链。
在另一个特定方面,提供了超激动性CD28抗原结合分子,其包含:两条各自包含SEQ ID NO:62的氨基酸序列的轻链、包含SEQ ID NO:83的氨基酸序列的第一重链和包含SEQ ID NO:84的氨基酸序列的第二重链。
在进一步方面,超激动性CD28抗原结合分子包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的VH和VL结构域,其中VH结构域经由肽连接基与Fc臼重链的C末端联结,并且其中VL结构域经由肽连接基与Fc杵重链的C末端联结。
在进一步方面,提供了如本文所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的crossFab片段,其经由肽连接基与所述两条重链中的一条的C末端联结。
对于与CD28结合为二价并且对于与肿瘤相关抗原结合为二价(2+2格式)的超激动性CD28抗原结合分子
在另一方面,提供了如本文所公开的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的两个crossFab片段,其中一个crossFab片段经由肽连接基与所述两条重链中的一条的C末端联结,并且其中另一个crossFab片段经由肽连接基与所述第二重链的C末端联结。
在一方面,如前文所述的超激动性CD28抗原结合分子包含两个能够与肿瘤相关抗原特异性结合的crossFab片段,其中一个crossFab片段经由肽连接基与两条重链中的一条的C末端联结,其中另一个crossFab片段经由肽连接基与第二重链的C末端联结,并且其中crossFab片段中的CH1和CL结构域被交换。在进一步方面,将crossFab片段各自在VH结构域的N末端与Fc结构域的C末端融合。
在一个特定方面,提供了超激动性CD28抗原结合分子,其包含:两条各自包含SEQID NO:65的氨基酸序列的轻链、两条各自包含SEQ ID NO:74的氨基酸序列的轻链和两条各自包含SEQ ID NO:73的氨基酸序列的重链。
在另一个特定方面,提供了超激动性CD28抗原结合分子,其包含:两条各自包含SEQ ID NO:65的氨基酸序列的轻链、两条各自包含SEQ ID NO:82的氨基酸序列的轻链和两条各自包含SEQ ID NO:81的氨基酸序列的重链。
对于与CD28结合为二价、对于与FAP结合为单价并且对于与CEA结合为单价的三特异性超激动性CD28抗原结合分子
在另一方面,提供了如本文所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,
(b)能够与FAP特异性结合的VH和VL结构域,其中VH结构域经由肽连接基与两条重链中的一条的C末端联结,并且其中VL结构域经由肽连接基与第二重链的C末端联结,和
(c)能够与CEA特异性结合的crossFab片段,其经由肽连接基与能够与FAP特异性结合的VH结构域的C末端联结。
在一个特定方面,提供了超激动性CD28抗原结合分子,其包含:两条各自包含SEQID NO:62的氨基酸序列的轻链、包含SEQ ID NO:109的氨基酸序列的轻链、包含SEQ ID NO:107的氨基酸序列的第一重链和包含SEQ ID NO:108的氨基酸序列的第二重链。
在另一方面,提供了如本文所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,
(b)能够与FAP特异性结合的VH和VL结构域,其中VH结构域经由肽连接基与两条重链中的一条的C末端联结,并且其中VL结构域经由肽连接基与第二重链的C末端联结,和
(c)能够与CEA特异性结合的VH和VL结构域,其中VH结构域经由肽连接基与能够与FAP特异性结合的VH结构域的C末端联结,并且其中VL结构域经由肽连接基与能够与FAP特异性结合的VL结构域的C末端联结。
在一个特定方面,提供了超激动性CD28抗原结合分子,其包含:两条各自包含SEQID NO:62的氨基酸序列的轻链、包含SEQ ID NO:110的氨基酸序列的第一重链和包含SEQID NO:111的氨基酸序列的第二重链。
降低Fc受体结合和/或效应子功能的Fc结构域修饰
本发明的超激动性CD28抗原结合分子的Fc结构域由一对包含免疫球蛋白分子的重链结构域的多肽链组成。例如,免疫球蛋白G(IgG)分子的Fc结构域是二聚体,该二聚体的每个亚基包含CH2和CH3 IgG重链恒定结构域。Fc结构域的两个亚基能够彼此稳定缔合。Fc结构域为本发明的抗原结合分子赋予有利的药代动力学特性,包括有助于靶组织中的良好积聚的长血清半衰期和有利的组织-血液分配比。另一方面,可能导致本发明的双特异性抗体不期望地靶向表达Fc受体的细胞,而不是优选的抗原携带细胞。
据此,与天然IgG1 Fc结构域相比,本发明的超激动性CD28抗原结合分子的Fc结构域表现出对Fc受体的结合亲和力降低和/或效应子功能的降低。在一方面,Fc基本上不结合Fc受体和/或不诱导效应子功能。在一个特定方面,Fc受体是Fcγ受体。在一个方面,Fc受体是人Fc受体。在一个具体方面,Fc受体是活化的人Fcγ受体,更具体地是人FcγRIIIa、FcγRI或FcγRIIa,最具体地是人FcγRIIIa。在一方面,Fc结构域不诱导效应子功能。降低的效应子功能可以包括但不限于以下中的一个或多个:降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。
在某些方面,一个或多个氨基酸修饰可引入本文提供的抗体的Fc区中,从而生成Fc区变体。Fc区变体可包含人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4 Fc区),其在一个或多个氨基酸位置上包含氨基酸修饰(例如取代)。
在一个特定方面,本发明提供了抗体,其中Fc区包含一个或多个氨基酸取代,该一个或多个氨基酸取代减少与Fc受体的结合,具体地是与Fcγ受体的结合。在一方面,本发明提供了抗体,其中Fc区包含一个或多个氨基酸取代,并且其中由抗体诱导的ADCC降低至由包含野生型人IgG1 Fc区的抗体诱导的ADCC的0-20%。
在一方面,抗体的Fc结构域包含降低Fc结构域对Fc受体的结合亲和力和/或效应子功能的一个或多个氨基酸突变。典型地,相同的一个或多个氨基酸突变存在于Fc结构域的两个亚基中的每一个中。特别地,Fc结构域在位置E233、L234、L235、N297、P331和P329(EU编号)处包含氨基酸取代。特别地,Fc结构域在IgG重链的位置234和235(EU编号)和/或329(EU编号)处包含氨基酸取代。更特别地,提供了根据本发明的抗体,其包含在IgG重链中具有氨基酸取代L234A、L235A和P329G(“P329G LALA”,EU编号)的Fc结构域。氨基酸取代L234A和L235A是指所谓的LALA突变。氨基酸取代的“P329G LALA”组合几乎完全消除了人IgG1 Fc结构域的Fcγ受体结合,并且在国际专利申请公开号WO 2012/130831 A1中有所描述,该公开也描述了制备此类突变型Fc结构域的方法和用于确定其特性诸如Fc受体结合或效应子功能的方法。
具有降低的Fc受体结合和/或效应子功能的Fc结构域还包括具有一个或多个Fc结构域残基238、265、269、270、297、327和329的取代的那些Fc结构域(美国专利号6,737,056)。此类Fc突变体包括在氨基酸位置265、269、270、297和327中的两处或更多处具有取代的Fc突变体,包括所谓的“DANA”Fc突变体,其残基265和297被取代为丙氨酸(美国专利号7,332,581)。
在另一方面,Fc结构域为IgG4 Fc结构域。与IgG1抗体相比,IgG4抗体表现出降低的对Fc受体的结合亲和力和降低的效应子功能。在一个更具体方面,Fc结构域是在位置S228处(Kabat编号)包含氨基酸取代(具体地是氨基酸取代S228P)的IgG4 Fc结构域。在一个更具体方面,Fc结构域是包含氨基酸取代L235E和S228P以及P329G(EU编号)的IgG4 Fc结构域。此类IgG4 Fc结构域突变体及其Fcγ受体结合特性也在WO 2012/130831中描述。
可以使用本领域熟知的遗传或化学方法,通过氨基酸缺失、取代、插入或修饰来制备突变Fc结构域。遗传方法可包括对编码DNA序列的位点特异性诱变、PCR、基因合成等。可以例如通过测序来验证正确的核苷酸变化。
与Fc受体的结合可以例如使用标准仪器(诸如BIAcore仪器(GE Healthcare),通过ELISA或通过表面等离子体共振(SPR)容易地确定,并且Fc受体诸如可以通过重组表达获得。另选地,可以使用已知表达特定Fc受体的细胞系(诸如表达FcγIIIa受体的人NK细胞)来评估Fc结构域或包含Fc结构域的细胞活化抗体对Fc受体的结合亲和力。
Fc结构域的效应子功能,或本发明的包含Fc结构域的抗原结合分子,可以通过本领域已知的方法来测量。本文描述了用于测量ADCC的合适测定法。用于评定目标分子的ADCC活性的体外测定的其他实例描述于美国专利号5,500,362;Hellstrom等人,Proc NatlAcad Sci USA 83,7059-7063(1986)和Hellstrom等人,Proc Natl Acad Sci USA 82,1499-1502(1985);美国专利号5,821,337;Bruggemann等人,J Exp Med 166,1351-1361(1987)。替代地,可以使用非放射性测定方法(参见例如,用于流式细胞术的ACTITM非放射性细胞毒性测定(CellTechnology,Inc.Mountain View,CA);以及CytoTox非放射性细胞毒性测定(Promega,Madison,WI))。用于此类测定的有用效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。替代地或另外地,可例如在诸如在Clynes等人,Proc NatlAcad Sci USA 95,652-656(1998)中公开的动物模型中体内评定目标分子的ADCC活性。
在一些方面,Fc结构域与补体组分,特别是C1q的结合减少。据此,在一些方面,其中Fc结构域经工程化改造以具有降低的效应子功能,所述降低的效应子功能包括降低的CDC。可以进行C1q结合测定以确定本发明的双特异性抗原结合分子是否能够结合C1q并因此具有CDC活性。参见例如WO 2006/029879和WO 2005/100402中的C1q和C3c结合ELISA。为了评定补体活化,可以执行CDC测定(参见例如Gazzano-Santoro等人,J Immunol Methods202,163(1996);Cragg等人,Blood 101,1045-1052(2003);以及Cragg和Glennie,Blood103,2738-2743(2004))。
在一个特定方面,与天然IgG1 Fc结构域相比表现出降低的对Fc受体的结合亲和力和/或降低的效应子功能的Fc结构域是包含氨基酸取代L234A、L235A和任选的P329G的人IgG1 Fc结构域,或包含氨基酸取代S228P、L235E和任选的P329G的人IgG4 Fc结构域(根据Kabat EU索引编号)。更特别地,它是包含氨基酸取代L234A、L235A和P329G(根据Kabat EU索引编号)的人IgG1 Fc结构域。
促进异源二聚化的Fc结构域修饰
本发明的超激动性CD28抗原结合分子包含与Fc结构域的两个亚基中的一个或另一个融合的不同抗原结合位点,因此该Fc结构域的两个亚基可包含在两个不相同的多肽链中。这些多肽的重组共表达和随后的二聚化导致了两种多肽的几种可能的组合。为了在重组生产中提高本发明的双特异性抗原结合分子的产率和纯度,因此在本发明的双特异性抗原结合分子的Fc结构域中引入促进所需多肽的缔合的修饰将是有利的。
据此,在特定方面,本发明涉及超激动性CD28抗原结合分子,其包含:(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,以及(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低抗原结分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代,其中Fc结构域包含促进Fc结构域的第一亚基和第二亚基的缔合的修饰。人IgG Fc结构域的两个亚基之间最广泛的蛋白质间相互作用位点在Fc结构域的CH3结构域中。因此,在一个方面,所述修饰在Fc结构域的CH3结构域中。
在一个具体方面,所述修饰是所谓的“突起进入孔”修饰,所述修饰包括Fc结构域的两个亚基中的一个中的“突起”修饰和Fc结构域的两个亚基中的另一个中的“孔”修饰。因此,本发明涉及超激动性CD28抗原结合分子,其包含:(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,以及(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低抗原结分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代,其中根据杵臼结构方法,Fc结构域的第一亚基包含杵,并且Fc结构域的第二亚基包含臼。在一个特定方面中,Fc结构域的第一亚基包含氨基酸取代S354C和T366W(EU编号),而Fc结构域的第二亚基包含氨基酸取代Y349C、T366S和Y407V(根据Kabat EU索引编号)。
杵臼结构技术描述于例如US 5,731,168;US 7,695,936;Ridgway等人,Prot Eng9,617-621(1996)和Carter,J Immunol Meth 248,7-15(2001)中。通常,该方法涉及在第一多肽的界面处引入凸起(“突起”)并在第二多肽的界面中引入相应的空腔(“孔”),使得所述凸起可以定位在所述空腔中,以便促进异二聚体的形成并阻碍同二聚体的形成。凸起是通过用较大侧链(例如酪氨酸或色氨酸)取代来自第一多肽的界面的小氨基酸侧链而构建的。具有与凸起相同或相似大小的补偿空腔是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在第二多肽的界面中创建的。
因此,在一个方面,在本发明的双特异性抗原结合分子的Fc结构域的第一亚基的CH3结构域中,某一氨基酸残基被具有较大侧链体积的氨基酸残基取代,从而在第一亚基的CH3结构域内产生凸起,所述凸起可定位于第二亚基的CH3结构域内的空腔中;而在Fc结构域的第二亚基的CH3结构域中,某一氨基酸残基被具有较小侧链体积的氨基酸残基取代,从而在第二亚基的CH3结构域内产生空腔,该第一亚基的CH3结构域内的所述凸起可定位在该空腔内。凸起和空腔可以通过改变编码多肽的核酸来制备,例如通过位点特异性诱变或通过肽合成。在一个具体方面,在Fc结构域的第一亚基的CH3结构域中,366位处的苏氨酸残基被色氨酸残基(T366W)取代,而在Fc结构域的第二亚基的CH3结构域中,407位处的酪氨酸残基被缬氨酸残基(Y407V)取代。在一个方面中,另外在Fc结构域的第二亚基中,366位处的苏氨酸残基被丝氨酸残基(T366S)取代,并且368位处的亮氨酸残基被丙氨酸残基(L368A)取代。
在另一个方面,另外在Fc结构域的第一亚基中,354位处的丝氨酸残基被半胱氨酸残基(S354C)取代,并且另外在Fc结构域的第二亚基中,349位的酪氨酸残基被半胱氨酸残基(Y349C)取代。引入这两个半胱氨酸残基导致在Fc结构域的两个亚基之间形成二硫桥,从而进一步稳定该二聚体(Carter(2001),J Immunol Methods 248,7-15)。在一个特定方面,Fc结构域的第一亚基包含氨基酸取代S354C和T366W(EU编号),而Fc结构域的第二亚基包含氨基酸取代Y349C、T366S和Y407V(根据Kabat EU索引编号)。
在另一方面,促进Fc结构域的第一亚基和第二亚基缔合的修饰包括介导静电转向效应的修饰,例如如在PCT公开WO 2009/089004中所述。通常,该方法涉及用带电荷的氨基酸残基取代两个Fc结构域亚基的界面处的一个或多个氨基酸残基,使得同源二聚体形成变得在静电上不利,但异源二聚化在静电上有利。
如本文报道的超激动性CD28抗原结合分子的重链的C末端可以是以氨基酸残基PGK结束的完整C末端。重链的C末端可以是缩短的C末端,在所述缩短的C末端中已经去除了一个或两个C末端氨基酸残基。在一个优选的方面,重链的C末端是以PG结束的缩短的C末端。在本文报道的所有方面中的一个方面,如本文所指定的包含含有C末端CH3结构域的重链的CD28抗原结合分子,包含C末端甘氨酸-赖氨酸二肽(G446和K447,根据Kabat EU索引编号)。在本文报道的所有方面中的一个方面,如本文所指定的包含含有C末端CH3结构域的重链的CD28抗原结合分子,包含C末端甘氨酸残基(G446,根据Kabat EU索引编号)。
Fab结构域中的修饰
一方面,本发明涉及超激动性CD28抗原结合分子,其包含:(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,以及(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低抗原结分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代,其中至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域是Fab片段,并且在该Fab片段中,可变结构域VH和VL或者恒定结构域CH1和CL根据Crossmab技术被交换。
在WO2009/080252和Schaefer,W.等人,PNAS,108(2011)11187-1191中详细描述了在一个结合臂中具有结构域置换/交换的多特异性抗体(CrossMabVH-VL或CrossMabCH-CL)。它们明显减少了由抗第一抗原的轻链与抗第二抗原的错误重链的错配导致的副产物(与没有此类结构域交换的方法相比)。
在一方面,本发明涉及超激动性CD28抗原结合分子,其包含:(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,(b)两个能够与肿瘤相关抗原特异性结合的抗原结合结构域,以及(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低抗原结分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代,其中在能够与肿瘤相关抗原特异性结合的Fab片段中,恒定结构域CL和CH1被彼此替换,使得CH1结构域是轻链的一部分并且CL结构域是重链的一部分。
在另一方面,并且为了进一步改善正确配对,包含以下各项的超激动性CD28抗原结合分子可含有不同的带电氨基酸取代(所谓“带电残基”):(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,(b)两个能够与肿瘤相关抗原特异性结合的抗原结合结构域,以及(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低抗原结分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代。将这些修饰引入交叉或非交叉的CH1和CL结构域中。在一个特定方面,本发明涉及超激动性CD28抗原结合分子,其中在CL结构域中的一个中,位置123(EU编号)处的氨基酸已被精氨酸(R)取代并且位置124(EU编号)处的氨基酸已被赖氨酸(K)取代;并且其中在CH1结构域中的一个中,位置147(EU编号)和位置213(EU编号)处的氨基酸已被谷氨酸(E)取代。在一个特定方面,在能够与CD28特异性结合的Fab片段的CL结构域中,位置123(EU编号)处的氨基酸已被精氨酸(R)取代并且位置124(EU编号)处的氨基酸已被赖氨酸(K)取代,并且在能够与CD28特异性结合的Fab片段的CH1结构域中,位置147(EU编号)和位置213(EU编号)处的氨基酸已被谷氨酸(E)取代。
多核苷酸
本发明进一步提供了编码如本文所述的超激动性CD28抗原结合分子或其片段的分离的多核苷酸。
编码本发明的超激动性CD28抗原结合分子的分离的多核苷酸可以表达为编码完整抗原结合分子的单个多核苷酸,或表达为共表达的多个(例如,两个或更多个)多核苷酸。由共表达的多核苷酸编码的多肽可以经由例如二硫键或其他手段缔合以形成功能性抗原结合分子。例如,免疫球蛋白的轻链部分可以由来自免疫球蛋白的重链部分的单独多核苷酸编码。当共表达时,重链多肽将与轻链多肽缔合以形成免疫球蛋白。
在一些方面,分离的多核苷酸编码如本文所述的根据本发明的整个超激动性CD28抗原结合分子。在其他方面,分离的多核苷酸编码包含在如本文所述的根据本发明的超激动性CD28抗原结合分子中的多肽。
在某些方面,多核苷酸或核酸是DNA。在其他方面,本发明的多核苷酸是RNA,例如以信使RNA(mRNA)的形式。本发明的RNA可以是单链或双链的。
重组方法
本发明的超激动性CD28抗原结合分子可以例如通过固态肽合成(例如Merrifield固相合成)或重组生产获得。对于重组生产,将例如如上所述的编码超激动性CD28抗原结合分子或其多肽片段的一种或多种多核苷酸分离并插入至一个或多个载体中以用于在宿主细胞中进一步克隆和/或表达。此类多核苷酸可使用常规方法容易地分离并且测序。在本发明的一个方面,提供了一种载体,优选为表达载体,该载体包含本发明的多核苷酸中的一种或多种多核苷酸。可以使用本领域技术人员熟知的方法来构建含有抗体(片段)的编码序列以及适当转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、合成技术和体内重组/遗传重组。参见例如以下文献所述的技术:Maniatis等人,MOLECULAR CLONING:ALABORATORY MANUAL,Cold Spring Harbor Laboratory,N.Y.(1989);和Ausubel等人,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Greene Publishing Associates and WileyInterscience,N.Y.(1989)。表达载体可以是质粒、病毒的一部分,或者可以是核酸片段。表达载体包括表达盒,编码抗体或其多肽片段的多核苷酸(即编码区)与启动子和/或其他转录或翻译控制元件可操作缔合地克隆至所述表达盒中。如本文所用,“编码区”是核酸的一部分,该部分由翻译成氨基酸的密码子组成。尽管“终止密码子”(TAG、TGA或TAA)未被翻译成氨基酸,但其(如果存在的话)可被认为是编码区的一部分,而任何侧翼序列,例如启动子、核糖体结合位点、转录终止子、内含子、5'和3'非翻译区等不是编码区的一部分。两个或更多个编码区可存在于单个多核苷酸构建体中(例如在单个载体上),或在单独的多核苷酸构建体中(例如在单独的(不同的)载体上)。此外,任何载体可含有单一编码区,或可包含两个或更多个编码区,例如本发明的载体可以编码一种或多种多肽,该一种或多种多肽在翻译后或翻译时通过蛋白水解切割分离成最终的蛋白质。此外,本发明的载体、多核苷酸或核酸可编码异源编码区,该异源编码区与编码本发明的抗体或其多肽片段的多核苷酸或其变体或衍生物融合或不融合。异源编码区包括但不限于特化元件或基序,诸如分泌信号肽或异源功能结构域。可操作缔合是当基因产物(例如多肽)的编码区以某种方式与一个或多个调控序列缔合,以使基因产物的表达处于调控序列的影响或控制下。如果启动子功能的诱导导致编码所需基因产物的mRNA的转录,并且如果两个DNA片段之间的连接性质不干扰表达调控序列指导基因产物表达的能力或干扰待转录的基因模板的能力,则该两个DNA片段(诸如多肽编码区和与其相关的启动子)是“可操作地缔合的”。因此,如果启动子能够影响该核酸的转录,则启动子区域将与编码多肽的核酸可操作地缔合。启动子可以是细胞特异性启动子,该细胞特异性启动子仅在预定细胞中指导DNA的实质转录。除启动子外,其他转录控制元件,例如增强子、操纵子、阻遏物和转录终止信号,可以与多核苷酸可操作地缔合以指导细胞特异性转录。
本文公开了合适的启动子和其他转录控制区。多种转录控制区是本领域技术人员已知的。这些转录控制区包括但不限于在脊椎动物细胞中起作用的转录控制区,诸如但不限于来自巨细胞病毒的启动子和增强子区段(例如立即早期启动子结合内含子-A)、猿猴病毒40(例如早期启动子)和逆转录病毒(诸如例如劳氏肉瘤病毒)。其他转录控制区包括来源于脊椎动物基因(诸如肌动蛋白、热休克蛋白、牛生长激素和兔珠蛋白)的那些转录控制区,以及能够控制真核细胞中基因表达的其他序列。其他合适的转录控制区包括组织特异性启动子和增强子以及诱导型启动子(例如四环素可诱导启动子)。类似地,各种翻译控制元件是本领域普通技术人员已知的。这些翻译控制元件包括但不限于核糖体结合位点、翻译起始和终止密码子,以及来源于病毒系统的元件(特别是内部核糖体进入位点,或IRES,也称为CITE序列)。表达盒还可以包括其他特征,诸如复制起点,和/或染色体整合元件,诸如逆转录病毒长末端重复序列(LTR),或腺相关病毒(AAV)反向末端重复序列(ITR)。
本发明的多核苷酸和核酸编码区可以与编码分泌肽或信号肽的附加编码区缔合,所述附加编码区指导由本发明的多核苷酸编码的多肽的分泌。例如,如果需要分泌抗体或其多肽片段,可以将编码信号序列的DNA置于本发明的核酸抗体或其多肽片段的上游。根据信号假设,由哺乳动物细胞分泌的蛋白质具有信号肽或分泌前导序列,一旦已经起始跨粗面内质网输出生长的蛋白质链,该信号肽或分泌前导序列就被从成熟蛋白质上切割下来。本领域普通技术人员知道由脊椎动物细胞分泌的多肽通常具有与所述多肽的N末端融合的信号肽,所述信号肽被从翻译的多肽上切割下来以产生所述多肽的分泌或“成熟”形式。在某些实施例中,使用天然信号肽(例如免疫球蛋白重链或轻链信号肽),或保留指导与其可操作地缔合的多肽分泌的能力的该序列的功能性衍生物。另选地,可以使用异源哺乳动物信号肽或其功能衍生物。例如,野生型前导序列可以被人组织纤溶酶原活化剂(TPA)或小鼠β葡糖醛酸酶的前导序列取代。
编码可用于促进后续纯化的短蛋白序列(例如,组氨酸标签)或帮助标记超激动性CD28抗原结合分子的DNA可包括在编码本发明的抗体或其多肽片段的多核苷酸的内部或末端。
在本发明的另一方面,提供了包含一种或多种本发明的多核苷酸的宿主细胞。在某些实施例中,提供了包含一种或多种本发明的载体的宿主细胞。多核苷酸和载体可以单独或组合地渗入本文中分别关于多核苷酸和载体描述的任何特征。在一方面,宿主细胞包含载体(例如,已使用载体转化或转染),该载体包含编码本发明的抗体(的一部分)的多核苷酸。如本文所用,术语“宿主细胞”是指可以被工程化改造以产生本发明的融合蛋白或其片段的任何种类的细胞系统。适于复制和支持抗原结合分子表达的宿主细胞是本领域中熟知的。此类细胞可以用特定的表达载体适当地转染或转导,并且可以生长大量含有载体的细胞以用于接种大规模发酵罐来获得足够量的抗原结合分子用于临床应用。合适的宿主细胞包括原核微生物,诸如大肠杆菌,或各种真核细胞,诸如中国仓鼠卵巢细胞(CHO)、昆虫细胞等。例如,多肽可以在细菌中产生,特别是当不需要糖基化时。多肽在表达后可以在可溶性级分中从细菌细胞糊状物中分离,并可以进一步纯化。除原核生物外,诸如丝状真菌或酵母之类的真核微生物也是用于编码多肽的载体的合适克隆或表达宿主,包括这样的真菌和酵母菌株,该真菌和酵母菌株的糖基化途径已经被“人源化”,从而导致产生具有部分或完全人糖基化模式的多肽。参见Gerngross,Nat Biotech 22,1409-1414(2004)和Li等人,NatBiotech 24,210-215(2006)。
用于表达(糖基化)多肽的合适宿主细胞还来源于多细胞生物(无脊椎动物和脊椎动物)。无脊椎动物细胞的实例包括植物细胞和昆虫细胞。已经鉴定出了可以与昆虫细胞结合使用,特别是用于转染草地夜蛾(Spodoptera frugiperda)细胞的许多杆状病毒株。植物细胞培养物也可用作宿主。参见例如美国专利号5,959,177、6,040,498、6,420,548、7,125,978和6,417,429(描述了用于在转基因植物中产生抗体的PLANTIBODIESTM技术)。脊椎动物细胞也可用作宿主。例如,适于在悬浮液中生长的哺乳动物细胞系可能是有用的。有用的哺乳动物宿主细胞系的其他实例为由SV40转化的猴肾CV1系(COS-7);人胚肾系(293或293T细胞,如例如在Graham等人,J Gen Virol 36,59(1977)中所述)、幼仓鼠肾细胞(BHK)、小鼠塞尔托利氏细胞(TM4细胞,如例如在Mather,Biol Reprod 23,243-251(1980)中所述)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝细胞(BRL 3A)、人肺细胞(W138)、人肝细胞(Hep G2)、小鼠乳腺肿瘤细胞(MMT060562)、TRI细胞(如例如在Mather等人,Annals N.Y.Acad Sci 383,44-68(1982)中所述)、MRC 5细胞,以及FS4细胞。其他有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞,包括dhfr-CHO细胞(Urlaub等人,Proc Natl Acad Sci USA 77,4216(1980));以及骨髓瘤细胞系,诸如YO、NS0、P3X63和Sp2/0。关于适用于蛋白质生产的某些哺乳动物宿主细胞系的综述,请参见例如Yazaki和Wu,Methods in Molecular Biology,第248卷(B.K.C.Lo编辑,Humana Press,Totowa,NJ),第255-268页(2003)。宿主细胞包括培养细胞,例如仅举几个例子而言哺乳动物培养细胞、酵母细胞、昆虫细胞、细菌细胞和植物细胞,还包括转基因动物、转基因植物或培养植物或动物组织中所包含的细胞。在一个实施例中,宿主细胞是真核细胞,优选哺乳动物细胞,诸如中国仓鼠卵巢(CHO)细胞、人胚肾(HEK)细胞或淋巴细胞(例如,Y0、NS0、Sp20细胞)。用于在这些系统中表达外源基因的标准技术是本领域已知的。可以对表达包含免疫球蛋白的重链或轻链的多肽的细胞进行工程化改造,以便也表达另一条免疫球蛋白链,使得表达的产物为具有重链和轻链的免疫球蛋白。
在一方面,提供了一种生产本发明的超激动性CD28抗原结合分子或其多肽片段的方法,其中所述方法包括在适于表达本发明的抗体或其多肽片段的条件下培养包含编码如本文提供的本发明的抗体或其多肽片段的多核苷酸的宿主细胞,以及从宿主细胞(或宿主细胞培养基)中回收本发明的抗体或其多肽片段。
在某些实施例中,能够与形成抗原结合分子的一部分的靶细胞抗原(例如,Fab片段)特异性结合的部分至少包含能够与抗原结合的免疫球蛋白可变区。可变区可以形成天然或非天然存在的抗体及其片段的一部分,并衍生自天然或非天然存在的抗体及其片段。产生多克隆抗体和单克隆抗体的方法是本领域熟知的(参见,例如,Harlow和Lane,"Antibodies,a laboratory manual",Cold Spring Harbor Laboratory,1988)。非天然存在的抗体可以使用固相肽合成来构建,可以重组产生(例如,如在美国专利号4,186,567中所述),或者可以例如通过筛选包含可变重链和可变轻链的组合文库来获得(参见例如McCafferty的美国专利号5,969,108)。
任何动物物种的免疫球蛋白均可用于本发明。可用于本发明的非限制性免疫球蛋白可以是鼠、灵长动物或人来源的。如果融合蛋白旨在用于人类使用,则可以使用嵌合形式的免疫球蛋白,其中该免疫球蛋白的恒定区来自人。也可以根据本领域熟知的方法来制备人源化或完全人形式的免疫球蛋白(参见,例如,Winter的美国专利号5,565,332)。人源化可通过各种方法实现,所述各种方法包括但不限于(a)将非人(例如供体抗体)CDR移植到人(例如受体抗体)架构和恒定区上,所述架构和恒定区有或没有保留关键架构残基(例如对于保持良好的抗原结合亲和力或抗体功能来说重要的关键架构残基),(b)仅将非人特异性决定区(SDR或a-CDR;对抗体-抗原相互作用至关重要的残基)移植到人架构和恒定区上,或者(c)移植整个非人可变结构域,但通过替换表面残基而用人样区段“隐藏”它们。人源化抗体及其制备方法在例如Almagro和Fransson,Front Biosci 13,1619-1633(2008)中综述,并进一步在例如Riechmann等人,Nature 332,323-329(1988);Queen等人,Proc Natl AcadSci USA 86,10029-10033(1989);美国专利号5,821,337、7,527,791、6,982,321和7,087,409;Jones等人,Nature 321,522-525(1986);Morrison等人,Proc Natl Acad Sci 81,6851-6855(1984);Morrison和Oi,Adv Immunol 44,65-92(1988);Verhoeyen等人,Science239,1534-1536(1988);Padlan,Molec Immun 31(3),169-217(1994);Kashmiri等人,Methods 36,25-34(2005)(描述SDR(a-CDR)移植);Padlan,Mol Immunol 28,489-498(1991)(描述“表面重整”);Dall’Acqua等人,Methods 36,43-60(2005)(描述“FR重排”);以及Osbourn等人,Methods 36,61-68(2005)和Klimka等人,Br J Cancer 83,252-260(2000)(描述用来进行FR重排的“导向选择”途径)中有所描述。根据本发明的特定免疫球蛋白是人免疫球蛋白。可以使用本领域已知的各种技术来产生人抗体和人可变区。人抗体一般描述于van Dijk和van de Winkel,Curr Opin Pharmacol 5,368-74(2001)和Lonberg,CurrOpin Immunol 20,450-459(2008)中。人可变区可以构成通过杂交瘤方法制备的人单克隆抗体的一部分并衍生自该人单克隆抗体(参见,例如,Monoclonal Antibody ProductionTechniques and Applications,pp.51-63(Marcel Dekker,Inc.,New York,1987))。还可以通过以下方式来制备人抗体和人可变区:将免疫原施用于转基因动物,所述转基因动物已被修饰以响应于抗原激发而产生具有人可变区的完整人抗体或完整抗体(参见,例如,Lonberg,Nat Biotech 23,1117-1125(2005))。还可以通过分离选自人源噬菌体展示文库的Fv克隆体可变区序列来产生人抗体和人可变区(参见,例如,Hoogenboom等人,在Methodsin Molecular Biology 178,1-37(O'Brien等人编,Human Press,Totowa,NJ,2001)中;和McCafferty等人,Nature 348,552-554;Clackson等人,Nature 352,624-628(1991))。噬菌体通常将抗体片段展示为单链Fv(scFv)片段或Fab片段。
在某些方面,根据例如PCT公开WO 2012/020006(参见与亲和力成熟有关的实例)或美国专利申请公开号2004/0132066中公开的方法,将超激动性CD28抗原结合分子工程化改造成具有增强的结合亲和力。本发明的抗原结合分子与特定抗原决定簇结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域技术人员熟悉的其他技术(例如,表面等离子体共振技术(Liljeblad等人,Glyco J 17,323-329(2000)),以及传统的结合测定法(Heeley,Endocr Res 28,217-229(2002))来测量。竞争测定法可用于鉴定与参考抗体竞争结合特定抗原的抗原结合分子。在某些实施例中,此类竞争抗原结合分子与由参考抗原结合分子结合的同一表位(例如,线性或构形表位)结合。用于定位抗原结合分子与之结合的表位的详细示例性方法提供于:Morris(1996),“Epitope Mapping Protocols”,出自Methods inMolecular Biology第66卷(Humana Press,Totowa,NJ)。在示例性竞争测定中,将固定化抗原在包含与抗原结合的第一经标记抗原结合分子和第二未标记抗原结合分子的溶液中孵育,该第二未标记抗原结合分子正在测试其与第一抗原结合分子竞争结合抗原的能力。第二抗原结合分子可以存在于杂交瘤上清液中。作为对照,将固定化抗原在包含第一个经标记抗原结合分子但不包含第二未标记抗原结合分子的溶液中孵育。在允许第一抗体与抗原结合的条件下孵育后,去除过量的未结合抗体,并测量与固定化抗原缔合的标记物的量。如果相对于对照样品,测试样品中与固定化抗原缔合的标记物的量大大减少,则表明第二抗原结合分子正在与第一抗原结合分子竞争与抗原的结合。参见Harlow和Lane(1988)Antibodies:A Laboratory Manual第14章(Cold Spring Harbor Laboratory,ColdSpring Harbor,NY)。
如本文所述制备的本发明的超激动性CD28抗原结合分子可通过本领域已知的技术纯化,这些技术为诸如高效液相色谱法、离子交换色谱法、凝胶电泳法、亲和色谱法、尺寸排阻色谱法等。用于纯化特定蛋白质的实际条件将部分取决于诸如净电荷、疏水性、亲水性等因素,并且对于本领域技术人员而言将是显而易见的。对于亲和色谱法纯化,可以使用抗原结合分子结合的抗体、配体、受体或抗原。例如,对于本发明的抗原结合分子的亲和色谱纯化,可以使用具有蛋白A或蛋白G的基质。基本上如实例中所述,可使用顺序蛋白A或G亲和色谱和尺寸排阻色谱来分离抗原结合分子。CD28抗原结合分子或其片段的纯度可以通过各种熟知的分析方法中任一者来确定,所述熟知的分析方法包括凝胶电泳法、高压液相色谱法等。例如,如实例中所述的被表达的CD28抗原结合分子被显示为完整的并且适当地组装,如还原和非还原SDS-PAGE所示。
测定
本文所提供的超激动性CD28抗原结合分子的物理/化学性质和/或生物学活性可通过本领域中已知的各种测定法进行鉴定、筛选或表征。
1.亲和力测定
本文提供的抗原结合分子对相应靶的亲和力可以使用标准仪器诸如Proteon仪器(Bio-rad)以及诸如可通过重组表达获得的受体或靶蛋白,根据实例中阐述的方法通过表面等离子体共振(SPR)来确定。含有TNF家族配体三聚物的抗原结合分子对靶细胞抗原的亲和力也可以使用标准仪器诸如Proteon仪器(Bio-rad)以及诸如可通过重组表达获得的受体或靶蛋白,通过表面等离子体共振(SPR)来确定。用于测量结合亲和力的特定说明性和示例性实施例描述于实例4中。根据一个方面,在25℃使用机器(Bio-Rad)通过表面等离子体共振来测量KD。
2.结合测定和其他测定
本文所提供的双特异性抗原结合分子与表达相应受体的细胞的结合可使用表达特定受体或靶抗原的细胞系例如通过流式细胞术(FACS)或通过表面等离子体共振(SPR)来评估。在一方面,在结合测定中使用表达人CD28的CHO细胞(经修饰以稳定地过表达人CD28的亲代细胞系CHO-k1 ATCC#CCL-61)。
在进一步方面,利用表达靶细胞抗原(例如FAP或CEA)的癌细胞系证明双特异性抗原结合分子与靶细胞抗原的结合。
3.活性测定
在一方面,提供了用于鉴定具有生物活性的CD28抗原结合分子的测定法。生物活性可以包括,例如,使用如实例5中所述的方法测量的T细胞增殖和细胞因子分泌或如实例6中测量的肿瘤细胞杀死。还提供了在体内和/或体外具有此类生物活性的抗体。
药物组合物、制剂和施用途径
在进一步方面,本发明提供了包含本文提供的超激动性CD28抗原结合分子中任一者的药物组合物,其例如用于以下治疗方法中的任一者中。在一个实施例中,药物组合物包含本文所提供的超激动性CD28抗原结合分子以及至少一种药用赋形剂。在另一实施例中,药物组合物包含本文提供的超激动性CD28抗原结合分子以及如下文所述的至少一种另外的治疗剂。
本发明的药物组合物包含治疗有效量的溶于或分散于药用赋形剂中的一种或多种抗原结合分子。术语“药学上或药理学上可接受的”是指分子实体和组合物在所采用的剂量和浓度下通常对接受者无毒,即当视情况而定施用于动物(例如人)时不产生不利的、过敏的或其他不良反应。含有至少一种超激动性CD28抗原结合分子和任选的另外的活性成分的药物组合物的制备将是鉴于本公开而为本领域的技术人员已知的,如由Remington'sPharmaceutical Sciences,第18版,Mack Printing Company,1990所示例的,该文献以引用方式并入本文。特别地,该组合物是冻干制剂或水溶液。如本文所用,“药用赋形剂”包括任何和所有溶剂、缓冲剂、分散介质、包衣、表面活性剂、抗氧化剂、防腐剂(例如抗细菌剂、抗真菌剂)、等渗剂、盐、稳定剂及它们的组合,如对本领域的普通技术人员所知。
肠胃外组合物包括设计用于注射(例如,皮下、皮内、病灶内、静脉内、动脉内、肌内、鞘内或腹膜内注射)的那些组合物。为了注射,可在水溶液中配制本发明的含TNF家族配体三聚物的抗原结合分子,优选在生理相容性缓冲液诸如Hanks溶液、林格氏溶液或生理盐水中配制。溶液可含有配制剂(formulatory agent),诸如悬浮剂、稳定剂和/或分散剂。替代地,超激动性CD28抗原结合分子可以是粉末形式,用于在使用前用合适的媒介物(例如,无菌无热原水)构建。根据需要,通过将本发明的融合蛋白以所需的量与下面列举的各种其他成分一起掺入适当的溶剂中,来制备无菌可注射溶液。例如,无菌可以通过无菌过滤膜过滤而容易地实现。通常,通过将各种灭菌的活性成分掺入含有基础分散介质和/或其他成分的无菌媒介物中来制备分散体。在用于制备无菌可注射溶液、悬浮液或乳液的无菌粉末的情况下,优选的制备方法是真空干燥或冻干技术,所述真空干燥或冻干技术产生来自先前无菌过滤的液体介质的活性成分加上任何附加所需成分的粉末。如果需要的话,液体介质应适当缓冲,并且在注射之前应首先使用足够的盐水或葡萄糖来使液体稀释剂等渗。该组合物必须是在制造和贮存条件下稳定的,并且保存为抗诸如细菌和真菌等微生物的污染作用。应当理解,内毒素污染应以例如低于0.5ng/mg蛋白质的安全水平保持最低。合适的药用赋形剂包括但不限于:缓冲剂,诸如磷酸盐、柠檬酸盐和其他有机酸;抗氧化剂,包括抗坏血酸和蛋氨酸;防腐剂(诸如十八烷基二甲基苄基氯化铵;氯化六甲双铵;苯扎氯铵;苄索氯铵;苯酚、丁醇或苄醇;对羟基苯甲酸烷基酯,诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯;儿茶酚;间苯二酚;环己醇;3-戊醇;间甲酚);低分子量(少于约10个残基)多肽;蛋白质,诸如血清白蛋白、明胶或免疫球蛋白;亲水性聚合物,诸如聚乙烯吡咯烷酮;氨基酸,诸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸;单糖、二糖和其他碳水化合物,包括葡萄糖、甘露糖或糊精;螯合剂,诸如EDTA;糖,诸如蔗糖、甘露醇、海藻糖或山梨糖醇;成盐抗衡离子,诸如钠;金属络合物(例如锌蛋白络合物);和/或非离子表面活性剂,诸如聚乙二醇(PEG)。水性注射悬浮液可含有增加悬浮液粘度的化合物,诸如羧甲基纤维素钠、山梨糖醇、葡聚糖等。任选地,悬浮液还可含有合适的稳定剂或增大化合物溶解度的试剂,以允许制备高浓度溶液。另外,活性化合物的悬浮液可以制备成适当的油性注射悬浮液。合适的亲脂性溶剂或载体包括脂肪油,诸如芝麻油;或合成脂肪酸酯,诸如油酸乙酯或甘油三酯;或脂质体。
活性成分可以包埋在例如通过凝聚技术或通过界面聚合而制备的微胶囊(例如分别为羟甲基纤维素或明胶微胶囊和聚(甲基丙烯酸甲酯)微胶囊)中;在胶体药物递送系统(例如,脂质体、白蛋白、微球、微乳液、纳米粒子和纳米胶囊)中;或在粗乳液中。此类技术在Remington's Pharmaceutical Sciences(第18版,Mack Printing Company,1990)中公开。可以制备缓释制备物。缓释制备物的合适示例包括含有多肽的固态疏水聚合物的半透性基质,所述基质是例如膜或微胶囊等成型制品的形式。在特定实施例中,可注射组合物的延长吸收可以通过在所述组合物中使用延迟吸收的试剂(例如单硬脂酸铝、明胶或它们的组合)来实现。
本文的示例性药用赋形剂还包括间质药物分散剂,诸如可溶中性活性透明质酸酶糖蛋白(sHASEGP),例如人可溶性PH-20透明质酸酶糖蛋白,诸如rHuPH20(Baxter International,Inc.)。某些示例性sHASEGP和使用方法,包括rHuPH20,描述于美国专利公开号2005/0260186和2006/0104968中。在一个方面中,将sHASEGP与一种或多种另外的糖胺聚糖酶(诸如软骨素酶)组合。
示例性的冻干抗体制剂描述于美国专利号6,267,958中。水性抗体制剂包括在美国专利号6,171,586和WO2006/044908中描述的那些,后一者中的制剂包含组氨酸-乙酸盐缓冲剂。
除了先前描述的组合物之外,超激动性CD28抗原结合分子也可以配制成长效制备物。此类长效制剂可以通过植入(例如皮下或肌内植入)或通过肌内注射施用。因此,例如,超激动性CD28抗原结合分子可以用合适的聚合或疏水材料配制(例如作为可接受油中的乳液)或用离子交换树脂配制,或配制为微溶的衍生物,例如配制为微溶盐。
包含本发明的超激动性CD28抗原结合分子的药物组合物可通过常规的混合、溶解、乳化、包封、包埋或冻干工艺进行生产。药物组合物可以使用一种或多种生理上可接受的载体、稀释剂、赋形剂或助剂以常规方式配制,所述载体、稀释剂、赋形剂或助剂有助于将蛋白质加工成可以在药学上使用的制备物。合适的制剂取决于所选择的施用途径。
本发明的超激动性CD28抗原结合分子可以配制成以游离酸或碱、中性或盐形式的组合物。药用盐是基本上保留游离酸或碱的生物活性的盐。这些药用盐包括酸加成盐,例如用蛋白质性质组合物的游离氨基形成的酸加成盐,或用无机酸(诸如盐酸或磷酸)或有机酸(诸如乙酸、草酸、酒石酸或扁桃酸)形成的酸加成盐。用游离羧基形成的盐也可以衍生自无机碱,诸如氢氧化钠、氢氧化钾、氢氧化铵、氢氧化钙或氢氧化铁;或者有机碱,诸如异丙胺、三甲胺、组氨酸或普鲁卡因。与相应的游离碱形式相比,药用盐倾向于更易溶于水性和其他质子溶剂中。
本文的组合物还可含有多于一种对于所治疗的特定适应症是必需的活性成分,优选是具有不会彼此不利地影响的互补活性的活性成分。此类活性成分适当地以对预期目的有效的量组合存在。
待用于体内施用的制剂通常是无菌的。例如,无菌可以通过无菌过滤膜过滤而容易地实现。
治疗方法和组合物
本文提供的超激动性CD28抗原结合分子中任一者可单独或联合用于治疗方法中。
在一方面,提供了用作药物的超激动性CD28抗原结合分子。在其他方面,提供了用于治疗癌症的超激动性CD28抗原结合分子。在某些方面,提供了用于治疗方法中的超激动性CD28抗原结合分子。在某些方面,本文提供了用于治疗患有癌症的个体的方法中的超激动性CD28抗原结合分子,该方法包括向个体施用治疗有效量的超激动性CD28抗原结合分子。在一个此类实施例中,该方法进一步包括向个体施用有效量的至少一种另外的治疗剂。
在进一步方面,提供了如本文所述的用于癌症免疫疗法的超激动性CD28抗原结合分子。在某些实施例中,提供了用于癌症免疫疗法的方法中的超激动性CD28抗原结合分子。根据任何上述方面的“个体”优选是人。
在进一步方面,本文提供了本文所述的超激动性CD28抗原结合分子在药物的制造或制备中的用途。在一个实施例中,该药物用于治疗癌症。在进一步方面,药物用于治疗癌症的方法中,该方法包括向患有癌症的个体施用有效量的该药物。在一个此类方面,该方法进一步包括向个体施用有效量的至少一种另外的治疗剂,例如,如下所述。根据上述方面中的任一方面的“个体”可以是人。
在进一步方面,本文提供了用于治疗癌症的方法。在一方面,该方法包括向患有癌症的个体施用有效量的超激动性CD28抗原结合分子。在一个此类方面,该方法进一步包括如下所述向个体施用有效量的至少一种另外的治疗剂。根据上述方面中的任一方面的“个体”可以是人。
在进一步方面,本文提供了包含本文所报导的超激动性CD28抗原结合分子中任一者的药物制剂,其例如用于上述治疗方法中的任一者中。在一方面,药物制剂包含本文所报导的超激动性CD28抗原结合分子中的任一者和药用载体。在另一方面,药物制剂包含本文所报导的超激动性CD28抗原结合分子中的任一者和至少一种另外的治疗剂。
本文所报导的抗体可以单独使用或与其他药剂联合用于治疗。例如,本文所报导的抗体可与至少一种另外的治疗剂共同施用。
上述此类组合疗法涵盖联合施用(其中两种或更多种治疗剂包括在相同或单独的制剂中)和单独施用,在单独施用的情况下,本文所报导的抗体的施用可以在施用另外的治疗剂或药剂之前、同时和/或之后进行。在一方面,超激动性CD28抗原结合分子的施用和另外的治疗剂的施用彼此在约一个月内或在约一周、两周或三周内或在约一天、两天、三天、四天、五天或六天内发生。
本文所报导的抗原结合分子(和任何另外的治疗剂)可以通过任何合适的方式施用,包括肠胃外、肺内和鼻内,并且如果需要的话用于局部治疗、病灶内施用。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。投配可以通过任何合适的途径进行,例如通过注射,诸如静脉内或皮下注射,部分取决于施用是短暂的还是长期的。本文考虑了各种给药方案,包括但不限于在各种时间点的单次或多次施用、团注施用,以及脉冲输注。
本文所述的超激动性CD28抗原结合分子将以与良好医学实践一致的方式配制、给药和施用。在这种情况下需要考虑的因素包括所治疗的特定疾患、所治疗的特定哺乳动物、个体患者的临床病症、疾患的原因、药剂的递送部位、施用方法、施用的时间安排,以及执业医师已知的其他因素。超激动性CD28抗原结合分子不必但可选地与一种或多种目前用于预防或治疗所讨论的疾患的制剂共同配制。此类其他药剂的有效量取决于制剂中存在的抗体的量、病症或治疗的类型,以及上面讨论的其他因素。这些通常以与本文所述相同的剂量和施用途径使用,或以本文所述剂量的约1%至99%使用,或以任何剂量且通过经验/临床上确定为合适的任何途径使用。
为了预防或治疗疾病,本文所述超激动性CD28抗原结合分子的适当剂量(当单独使用或与一种或多种其他的另外的治疗剂联合使用时)将取决于待治疗的疾病类型、抗体的类型、疾病的严重程度和病程、施用抗体用于预防还是治疗目的、患者的病史和对抗体的应答以及主治医师的酌处权。超激动性CD28抗原结合分子适当地一次或在一系列治疗中施用于患者。取决于疾病的类型和严重程度,约1μg/kg至15mg/kg(例如0.5mg/kg-10mg/kg)的超激动性CD28抗原结合分子可以是例如通过一次或多次单独施用或通过连续输注而施用于患者的初始候选剂量。取决于上述因素,一种典型的日剂量的范围可以为约1μg/kg至100mg/kg或更多。对于数天或更长时间的重复施用,取决于病症,治疗通常会持续直至发生所需的疾病症状抑制。抗体的一种示例性剂量的范围为约0.05mg/kg至约10mg/kg。因此,可以向患者施用约0.5mg/kg、2.0mg/kg、4.0mg/kg或10mg/kg(或它们的任何组合)的一种或多种剂量。此类剂量可以间歇施用,例如,每周或每三周施用(例如,使得患者接受约两次至约二十次,或例如约六次剂量的抗体)。可施用初始较高负荷剂量,然后施用一种或多种较低剂量。然而,其他剂量方案可能有用。该疗法的进展通过常规技术和测定而容易地监测。
其他药剂和治疗
本发明的超激动性CD28抗原结合分子可在治疗中与一种或多种其他药剂联合施用。例如,本发明的抗原结合分子可与至少一种另外的治疗剂共同施用。术语“治疗剂”包括可以施用用于治疗需要此类治疗的个体的症状或疾病的任何药剂。此类附加治疗剂可包含适合于所治疗的具体适应症的任何活性成分,优选地是具有不会彼此不利地影响的互补活性的活性成分。在某些实施例中,附加治疗剂是另一种抗癌剂。
此类其他药剂适当地以对预期目的有效的量组合存在。此类其他药剂的有效量取决于所用抗原结合分子的量、病症或治疗的类型,以及上面讨论的其他因素。超激动性CD28抗原结合分子通常以与本文所述相同的剂量和施用途径使用,或以本文所述剂量的约1%至99%使用,或以经验上/临床上确定为合适的任何剂量和任何途径使用。
上述此类联合疗法包括联合施用(其中两种或更多种治疗剂包括在相同或不同的组合物中)和单独施用,在单独施用的情况下,本发明的超激动性CD28抗原结合分子的施用可以在施用另外的治疗剂和/或佐剂之前、同时和/或之后进行。
制品
在本发明的另一个方面中,提供了一种制品,其含有可用于治疗、预防和/或诊断上述病症的物质。该制品包括容器和在所述容器上或与所述容器相关的标签或包装插页(package insert)。合适的容器包括例如瓶子、小瓶、注射器、静脉注射(IV)溶液袋等。所述容器可以由诸如玻璃或塑料等多种材料形成。容器容纳组合物,该组合物单独或与另一种有效用于治疗、预防和/或诊断所述病症的组合物组合,并且容器可具有无菌入口(例如容器可以是静脉注射溶液袋或具有可用皮下注射针刺穿的塞子的小瓶)。组合物中的至少一种活性剂是本发明的超激动性CD28抗原结合分子。
标签或包装插页指示该组合物用于治疗所选择的病症。此外,制品可包括(a)含有容纳在其中的组合物的第一容器,其中该组合物包含本发明的超激动性CD28抗原结合分子;以及(b)含有容纳在其中的组合物的第二容器,其中该组合物包含进一步的细胞毒性剂或其他治疗剂。本发明这一实施例中的制品进一步可包含包装插页,所述包装插页指示所述组合物可用于治疗特定病症。
另选地或另外地,制品还可包括第二(或第三)容器,该二(或第三)容器包括药用缓冲液,诸如抑菌性注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液和葡萄糖溶液。制品还可以包括从商业和用户角度所需的其他物质,包括其他缓冲剂、稀释剂、过滤器、针头和注射器。
表B(序列):
关于人免疫球蛋白轻链和重链的核苷酸序列的一般信息给出于:Kabat,E.A.等人,Sequences of Proteins of Immunological Interest,第5版,Public HealthService,National Institutes of Health,Bethesda,MD(1991)中。根据如上所定义的根据Kabat(Kabat,E.A.等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991))的编号系统来对抗体链的氨基酸进行编号和引用。
以下编号的段落描述了本发明的各方面:
1.一种超激动性CD28抗原结合分子,其能够与CD28二价结合并且包含
(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,
(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,和
(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代。
2.根据段落1所述的超激动性CD28抗原结合分子,其包含两个能够与CD28特异性结合的抗原结合结构域。
3.根据段落1或2所述的超激动性CD28抗原结合分子,其中Fc结构域是IgG,特别是IgG1 Fc结构域或IgG4 Fc结构域。
4.根据段落1至3中任一项所述的超激动性CD28抗原结合分子,其中Fc结构域属于人IgG1亚类并且包含氨基酸突变L234A、L235A和P329G(根据Kabat EU索引编号)。
5.根据段落1至4中任一项所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(i)重链可变区(VHCD28),其包含SEQ ID NO:20的重链互补决定区CDR-H1、SEQ IDNO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的轻链互补决定区CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3;或
(ii)重链可变区(VHCD28),其包含SEQ ID NO:36的CDR-H1、SEQ ID NO:37的CDR-H2和SEQ ID NO:38的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:39的CDR-L1、SEQ ID NO:40的CDR-L2和SEQ ID NO:41的CDR-L3。
6.根据段落1至5中任一项所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQ ID NO:20的CDR-H1、SEQ ID NO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3。
7.根据段落1至5中任一项所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含与SEQ IDNO:26的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCD28),其包含与SEQ ID NO:27的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
8.根据段落1至5中任一项所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含选自由SEQID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQID NO:48、SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51组成的组的氨基酸序列;以及轻链可变区(VLCD28),其包含选自由SEQ ID NO:27、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ IDNO:60和SEQ ID NO:60组成的组的氨基酸序列。
9.根据段落1至5或段落8中任一项所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(a)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(b)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(c)重链可变区(VHCD28),其包含SEQ ID NO:51的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:61的氨基酸序列,或
(d)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(e)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(f)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(g)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(h)重链可变区(VHCD28),其包含SEQ ID NO:43的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(i)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(j)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(k)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列。
10.根据段落1至9中任一项所述的超激动性CD28抗原结合分子,其中能够与CD28特异性结合的抗原结合结构域中的每一个均为Fab片段。
11.根据段落1至10中任一项所述的超激动性CD28抗原结合分子,其中能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与癌胚抗原(CEA)特异性结合的抗原结合结构域。
12.根据段落1至12中任一项所述的超激动性CD28抗原结合分子,其中能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含:(i)CDR-H1,其包含SEQID NO:127的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:128的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:129的氨基酸序列;以及轻链可变区(VLCEA),其包含:(iv)CDR-L1,其包含SEQ ID NO:130的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:131的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:132的氨基酸序列。
13.根据段落1至12中任一项所述的超激动性CD28抗原结合分子,其中能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含与SEQ ID NO:133的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCEA),其包含与SEQ ID NO:134的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
14.根据段落1至10中任一项所述的超激动性CD28抗原结合分子,其中能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与成纤维细胞活化蛋白(FAP)特异性结合的抗原结合结构域。
15.根据段落1至10或段落14中任一项所述的超激动性CD28抗原结合分子,其中能够与FAP特异性结合的抗原结合结构域包含
(a)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:12的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:13的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:14的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:15的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:16的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:17的氨基酸序列;或
(b)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:4的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:5的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:6的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:7的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:8的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:9的氨基酸序列。
16.根据段落1至10或段落14或15中任一项所述的超激动性CD28抗原结合分子,其中能够与FAP特异性结合的抗原结合结构域包含
(a)重链可变区(VHFAP),其包含与SEQ ID NO:18的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ IDNO:19的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;或
(b)重链可变区(VHFAP),其包含与SEQ ID NO:10的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ IDNO:11的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
17.根据段落1至10或段落14至16中任一项所述的超激动性CD28抗原结合分子,其中能够与FAP特异性结合到的抗原结合结构域包含:重链可变区(VHFAP),其包含SEQ IDNO:18的氨基酸序列;以及轻链可变区(VLFAP),其包含SEQ ID NO:19的氨基酸序列。
18.根据段落1至17中任一项所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的VH和VL结构域,其中所述VH结构域经由肽连接基与两条重链中的一条的C末端联结,并且其中所述VL结构域经由肽连接基与第二重链的C末端联结。
19.根据段落1至17中任一项所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的crossFab片段,其经由肽连接基与两条重链中的一条的C末端联结。
20.根据段落1至17中任一项所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和包含一个或多个降低抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的两个crossFab片段,其中一个crossFab片段经由肽连接基与两条重链中的一条的C末端联结,并且其中另一个crossFab片段经由肽连接基与第二重链的C末端联结。
21.一种多核苷酸,其编码根据段落1至20中任一项所述的双特异性抗原结合分子。
22.一种宿主细胞,其包含根据段落21所述的多核苷酸。
23.一种生产根据段落1至20中任一项所述的超激动性CD28抗原结合分子的方法,该方法包括在适于表达双特异性抗原结合分子的条件下培养根据段落22所述的宿主细胞。
24.一种药物组合物,其包含根据段落1至20中任一项所述的超激动性CD28抗原结合分子以及至少一种药用赋形剂。
25.根据段落24所述的药物组合物,用于治疗癌症。
26.根据段落1至20中任一项所述的超激动性CD28抗原结合分子或根据段落24所述的药物组合物,其用作药物。
27.根据段落1至20中任一项所述的超激动性CD28抗原结合分子或根据段落24所述的药物组合物,其用于治疗癌症。
28.根据段落1至20中任一项所述的用于治疗癌症的超激动性CD28抗原结合分子,其中超激动性CD28抗原结合分子与化疗剂、放疗和/或用于癌症免疫疗法的其他药剂联合施用。
29.根据段落1至20中任一项所述的超激动性CD28抗原结合分子或根据段落24所述的药物组合物在制造用于治疗癌症的药物中的用途。
30.一种抑制个体中肿瘤细胞生长的方法,该方法包括向个体施用有效量的根据段落1至20中任一项所述的超激动性CD28抗原结合分子或根据段落24所述的药物组合物,以抑制肿瘤细胞的生长。
31.一种治疗癌症的方法,该方法包括向个体施用治疗有效量的根据权利要求1至20中任一项所述的超激动性CD28抗原结合分子或根据权利要求24所述的药物组合物。
***
实例
以下是本发明的方法和组合物的实施例。应当理解,在给出以上提供的一般描述的情况下,可以实践各种其他实施例。
重组DNA技术
使用标准方法来操纵DNA,如在Sambrook等人,Molecular cloning:A laboratorymanual;Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York,1989中所述。根据制造商的说明来使用分子生物学试剂。关于人免疫球蛋白轻链和重链的核苷酸序列的一般信息在以下参考文献中给出:Kabat,E.A.等人,(1991)Sequences of Proteinsof Immunological Interest,第五版,NIH Publication No 91-3242。
DNA测序
通过双链测序确定DNA序列。
基因合成
若需要,通过使用适当模板进行PCR生成所需的基因区段,或由Geneart AG(Regensburg,Germany)或Genscript(New Jersey,USA)通过自动化基因合成从合成寡核苷酸和PCR产物合成所需的基因区段。将侧接有单个限制性内切酶切割位点的基因区段克隆到标准克隆/测序载体中。从转化的细菌中纯化出质粒DNA,并通过紫外光谱法确定浓度。通过DNA测序来确认亚克隆基因片段的DNA序列。设计具有合适限制性位点的基因区段,以允许亚克隆到相应的表达载体中。所有构建体均设计有5’端DNA序列,该序列编码前导肽,该前导肽靶向真核细胞分泌的蛋白。
细胞培养技术
使用如在Current Protocols in Cell Biology(2000),Bonifacino,J.S.,Dasso,M.,Harford,J.B.,Lippincott-Schwartz,J.和Yamada,K.M.(编辑),John Wiley&Sons,Inc中所述的标准细胞培养技术。
蛋白质纯化
参照标准方案从过滤的细胞培养物上清液中纯化蛋白质。简言之,将抗体施加至蛋白A琼脂糖柱(GE healthcare)并用PBS洗涤。在pH 2.8下实现抗体的洗脱,之后立即中和样品。通过尺寸排阻色谱法(Superdex 200,GE Healthcare)在PBS中或在20mM组氨酸、150mM NaCl pH 6.0中将聚集的蛋白与单体抗体分离。将单体抗体级分合并,使用例如MILLIPORE Amicon Ultra(30MWCO)离心浓缩器浓缩(若需要),冷冻并储存在-20℃或-80℃下。提供样品的部分以例如通过SDS-PAGE、尺寸排阻色谱(SEC)或质谱法来进行后续的蛋白质分析和分析表征。
SDS-PAGE
根据制造商的说明使用预制凝胶系统(Invitrogen)。具体地,使用10%或4-12%Bis-TRIS预制凝胶(pH 6.4)和MES(还原凝胶,具有抗氧化剂电泳缓冲添加剂)或MOPS(非还原凝胶)电泳缓冲液。
分析型尺寸排阻色谱
用于测定抗体的聚集和低聚物状态的尺寸排阻色谱法(SEC)通过HPLC色谱法进行。简而言之,将蛋白A纯化的抗体施加至Agilent HPLC 1100系统上的300mM NaCl、50mMKH2PO4/K2HPO4,pH 7.5中的Tosoh TSKgel G3000SW柱,或施加至Dionex HPLC系统上的2×PBS中的Superdex 200柱(GE Healthcare)。通过UV吸光度和峰面积积分来定量洗脱的蛋白质。将BioRad凝胶过滤标准品151-1901用作标准品。
质谱
本节描述了对具有VH/VL交换(VH/VL CrossMab)的多特异性抗体的表征,重点在于所述多特异性抗体的正确装配。通过对脱糖基化的完整CrossMab和脱糖基化/纤溶酶消化或替代地脱糖基化/限制性LysC消化的CrossMab进行电喷雾电离质谱(ESI-MS),来分析预期的一级结构。
在37℃下以1mg/ml的蛋白质浓度将VH/VL CrossMab用磷酸盐或Tris缓冲液中的N-糖苷酶F脱糖基化至多17h。纤溶酶消化或限制性LysC(Roche)消化用pH 8的Tris缓冲液中的100μg脱糖基化的VH/VL CrossMab分别在室温执行120小时和在37℃执行40min。在质谱分析之前,将样品经由HPLC在Sephadex G25柱(GE Healthcare)上脱盐。在配备有TriVersa NanoMate源(Advion)的maXis 4G UHR-QTOF MS系统(Bruker Daltonik)上经由ESI-MS来测定总质量。
使用表面等离子体共振(SPR)测定多特异性抗体对相应抗原的结合亲和力(BIACORE)
通过使用BIACORE仪器(GE Healthcare Biosciences AB,Uppsala,Sweden)进行表面等离子体共振来研究产生的抗体与相应抗原的结合。简而言之,对于亲和力测量,将山羊抗人IgG、JIR 109-005-098抗体通过胺偶联固定在CM5芯片上,以呈递针对相应抗原的抗体。在HBS缓冲液(HBS-P(10mM HEPES、150mM NaCl、0.005%吐温20,ph 7.4)中在25℃(或替代地在37℃)测量结合。在溶液中以各种浓度添加抗原(R&D Systems或经内部纯化)。通过80秒至3分钟的抗原注射来测量缔合;通过用HBS缓冲液洗涤芯片表面3至10分钟来测量解离,并使用1:1Langmuir结合模型估算KD值。从样品曲线中减去阴性对照数据(例如,缓冲液曲线),以校正系统固有基线漂移并降低噪声信号。使用相应的Biacore评估软件来分析传感图和计算亲和力数据。
实例1
靶向CD28和成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)的双特异性或三特异性抗体的生成和生产
1.1靶向CD28和成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)的双特异性或三特异性抗体的克隆
抗原的克隆:
将编码人CD28(Uniprot:P10747)的细胞外结构域(成熟蛋白质的氨基酸1至134)的DNA片段在框架中插入位于编码人IgG1 Fc片段(其用作溶解和纯化标签)的片段上游的两种不同哺乳动物接纳载体中。一种表达载体在Fc区包含“臼”突变,另一种则包含“杵”突变以及C末端avi标签(GLNDIFEAQKIEWHE,SEQ ID NO:162),可在与Bir A生物素连接酶共表达时进行特异性生物素化反应。另外,两个Fc片段均包含PG-LALA突变。将两种载体与编码BirA生物素连接酶的质粒联合转染,以得到在Fc-杵链的C末端具有单价生物素化的avi标签的二聚CD28-Fc构建体。
FAP克隆体4B9的可变结构域、CEA结合剂以及CD28克隆体SA和mAb 9.3用于生成多种靶向肿瘤的CD28构建体。FAP克隆体4B9的形成和制备在WO 2012/020006 A2中公开,该专利以引用方式并入本文。WO 2007/071422中描述了本文中称为MEDI-565的CEA克隆体,WO2006/050949中描述了CD28超激动性抗体(SA)。抗体mAb 9.3的描述可以在Tan等人.J.Immunology 2002,169,1119-1125中找到。为了生成相应的表达质粒,使用相应可变结构域的序列,并与预先插入相应接纳者哺乳动物表达载体中的相应恒定区在框内亚克隆。在图1A至1L中示出了所得分子的示意图。在指出之处,已经将Pro329Gly、Leu234Ala和Leu235Ala突变(PG-LALA)引入人IgG1重链的恒定区中,以消除与Fcγ受体的结合。为了生成不对称的双特异性抗体,Fc片段包含“杵”或“臼”突变,以避免重链错配。为了避免双特异性和多特异性抗体构建体中轻链的错配,将VH/VL或CH1/Cκ结构域的交换引入一个结合部分中(CrossFab技术)。在另一个结合部分中,将电荷引入CH1和Cκ结构域中。
克隆了以下分子,特定分子的示意图显示在图1A至1L中:
分子A:CD28(SA)(hu IgG4),TGN1412,人IgG4同种型中的CD28(SA)抗体(图1A),包含SEQ ID NO:62和SEQ ID NO:63的氨基酸序列(P1AE1975)。
分子B:CD28(SA)(PG-LALA),huIgG1 PG-LALA同种型中的CD28(SA)抗体(图1B),包含SEQ ID NO:62和SEQ ID NO:64的氨基酸序列(P1AD9289)。
分子C:FAP(4B9)-CD28(SA)1+1格式,双特异性huIgG1 PG-LALA CrossFab分子,具有CD28(SA)Fab片段(杵)中的带电修饰和FAP(4B9)Fab片段(臼)中的VH/VL交换(图1C),包含SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67和SEQ ID NO:68的氨基酸序列(P1AD4492)。
分子D:FAP(4B9)-CD28(SA)1+4格式,双特异性四价抗CD28(SA)和单价抗FAPhuIgG1 PG-LALA构建体。FAP克隆体4B9的VH和VL结构域融合至Fc结构域的相应链(VH:杵链;VL:臼链)的C末端(图1F)。该分子包含SEQ ID NO:62、SEQ ID NO:69和SEQ ID NO:70的氨基酸序列(P1AD9018)。
分子E:FAP(4B9)-CD28(SA)1+2格式,双特异性二价抗CD28(SA)和单价抗FAPhuIgG1 PG-LALA构建体。FAP克隆体4B9的VH和VL结构域融合至Fc结构域的相应链(VH:杵链;VL:臼链)的C末端(图1D)。该分子包含SEQ ID NO:62、SEQ ID NO:71和SEQ ID NO:72的氨基酸序列(P1AD9011)。
分子F:FAP(4B9)-CD28(SA)2+2,双特异性二价抗CD28(SA)和二价抗FAP huIgG1PG-LALA CrossFab构建体,抗CD28 Fab片段中具有带电修饰,具有CH1/Cκ交换的抗FAPCrossFab片段VH融合至Fc片段的C末端(图1E)。该分子包含SEQ ID NO:65、SEQ ID NO:73和SEQ ID NO:74的氨基酸序列(P1AD4493)。
分子G:FAP(4B9)-CD28(SA)2+1,双特异性单价抗CD28(SA)和二价抗FAP huIgG1PG-LALA CrossFab构建体,“经典取向”,抗CD28 CrossFab片段中具有VH/VL交换,抗FAPFab片段中具有带电修饰。该分子包含SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77和SEQID NO:78的氨基酸序列(P1AD5231)。
分子H:FAP(4B9)-CD28(SA)C-01,1+1双特异性单价抗CD28(SA)和单价抗FAPhuIgG1 PG-LALA CrossFab分子,“头至尾”,抗CD28 CrossFab片段中具有VH/VL交换,抗FAP结合剂中具有带电修饰。该分子包含SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:78和SEQ IDNO:79的氨基酸序列(P1AE2021)。
分子I:FAP(4B9)-CD28(SA)C-04,1+1双特异性单价抗CD28(SA)和单价抗FAPhuIgG1 PG-LALA构建体。FAP结合剂4B9的VH和VL结构域融合至Fc片段的相应链(VH:杵链;VL:臼链)的C末端。该分子包含SEQ ID NO:62、SEQ ID NO:72和SEQ ID NO:80的氨基酸序列(P1AE2236)。
分子J:CEA(Medi565)-CD28SA)2+2,双特异性二价抗CD28(SA)和二价抗CEAhuIgG1 PG-LALA CrossFab构建体,抗CD28 Fab片段中具有带电修饰,具有CH1/Cκ交换的抗CEA CrossFab片段VH融合至Fc片段的C末端(图1H)。该分子包含SEQ ID NO:65、SEQ ID NO:81和SEQ ID NO:82的氨基酸序列(P1AE1195)。
分子K:CEA(Medi565)-CD28(SA)1+2,双特异性二价抗CD28(SA)和单价抗CEAhuIgG1 PG-LALA构建体。CEA结合剂的VH和VL结构域融合至Fc片段的相应链(VH:杵链;VL:臼链)的C末端(图1G)。该分子包含SEQ ID NO:62、SEQ ID NO:83和SEQ ID NO:84的氨基酸序列(P1AE1194)。
分子L:单价IgG CD28(SA),单价抗单价抗CD28(SA)huIgG1 PG-LALA构建体,其中CD28被表达为与Fc(杵)片段组合的“臼”Fc链(图1I)。该分子包含SEQ ID NO:65、SEQ IDNO:85和SEQ ID NO:86的氨基酸序列(P1AD8944)。
分子M:CEA-CD28(SA)1+1格式,双特异性huIgG1 PG-LALA CrossFab分子,具有CD28(SA)Fab片段(杵)中的带电修饰和CEA crossFab片段(臼)中的VH/VL交换(图1J),包含SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:87和SEQ ID NO:88的氨基酸序列(P1AE3127)。
分子N:mab 9.3(PG-LALA),人IgG1 PG-LALA同种型的mAb9.3克隆体(如图1B所示)。该分子包含SEQ ID NO:89和SEQ ID NO:90的氨基酸序列(P1AD5142)。
分子O:FAP(4B9)-CD28(mAb9.3)C-03,双特异性huIgG1 PG-LALA CrossFab构建体,具有mAb9.3 Fab片段(杵)中的带电修饰和抗Fab片段(臼)中的VH/VL交换(如图1C所示)。该分子包含SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:91和SEQ ID NO:92的氨基酸序列(P1AE2238)。
分子P:FAP(4B9)-CD28(mAb9.3)1+4,双特异性四价抗CD28 mAb9.3和抗FAPhuIgG1 PG-LALA构建体。FAP结合剂的VH和VL结构域融合至Fc片段的相应链(VH:杵链;VL:臼链)的C末端(如图1F所示)。该分子包含SEQ ID NO:89、SEQ ID NO:93和SEQ ID NO:94的氨基酸序列(P1AD8969)。
分子Q:FAP(4B9)-CD28(mAb9.3)1+2,双特异性二价抗CD28 mAb9.3和单价抗FAPhuIgG1 PG-LALA构建体。FAP结合剂的VH和VL结构域融合至Fc片段的相应链(VH:杵链;VL:臼链)的C末端(如图1D所示)。该分子包含SEQ ID NO:89、SEQ ID NO:95和SEQ ID NO:96的氨基酸序列(P1AD8962)。
分子R:FAP(4B9)-CD28(mAb9.3)2+2,双特异性二价抗CD28(mAb9.3)和二价抗FAPhuIgG1 PG-LALA CrossFab构建体,mAb9.3 Fab片段中具有带电修饰,具有CH1/CκCrossFab交换的抗FAP Fab片段VH融合至Fc片段的C末端(如图1E所示)。该分子包含SEQ ID NO:97、SEQ ID NO:98和SEQ ID NO:99的氨基酸序列(P1AD8968)。
分子S:FAP(4B9)-CD28(mAb9.3)2+1,双特异性单价抗CD28(mAb9.3)和二价抗FAPhuIgG1 PG-LALA CrossFab构建体,“经典取向”,抗CD28(mAb9.3)CrossFab片段中具有VH/VL交换,抗FAP Fab片段中具有带电修饰。该分子包含SEQ ID NO:76、SEQ ID NO:77、SEQ IDNO:100和SEQ ID NO:101的氨基酸序列(P1AD5560)。
分子T:FAP(4B9)-CD28(mAb9.3)C-02,双特异性单价抗CD28(mAb9.3)和单价抗FAPhuIgG1 PG-LALA CrossFab构建体,“头至尾”,抗CD28(mAb9.3)CrossFab片段中具有VH/VL交换,抗FAP片段中具有带电修饰。该分子包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:100和SEQ ID NO:101的氨基酸序列(P1AE2022)。
分子U:FAP(4B9)-CD28(mAb9.3)C-05,双特异性单价抗CD28(mAb9.3)和单价抗FAPhuIgG1 PG-LALA构建体。FAP结合剂4B9的VH和VL结构域融合至Fc片段的相应链的C末端(VH:Fc杵链;VL:Fc臼链)。该分子包含SEQ ID NO:80、SEQ ID NO:89和SEQ ID NO:96的氨基酸序列(P1AE2237)。
分子V:CEA-CD28(mAb9.3)2+2,双特异性二价抗CD28(mAb9.3)和二价抗CEAhuIgG1 PG-LALA CrossFab构建体,mAb9.3 Fab片段中具有带电修饰,具有CH1/Cκ交换的抗CEA CrossFab片段VH融合至Fc片段的C末端(如图1H所示)。该分子包含SEQ ID NO:82、SEQID NO:89和SEQ ID NO:102的氨基酸序列(P1AE1193)。
分子W:CEA-CD28(mAb9.3)1+2,双特异性二价抗CD28(mAb9.3)和单价抗CEAhuIgG1 PG-LALA构建体。CEA结合剂的VH和VL结构域融合至Fc片段的相应链(VH:杵链;VL:臼链)的C末端(如图1G所示)。该分子包含SEQ ID NO:89、SEQ ID NO:103和SEQ ID NO:104的氨基酸序列(P1AE1192)。
分子X:单价IgG CD28(mAb9.3),其中CD28重链被表达为与Fc(杵)片段组合的“臼”Fc链(如图1I中所示)。该分子包含SEQ ID NO:86、SEQ ID NO:105和SEQ ID NO:106的氨基酸序列(P1AD8938)。
分子Y:FAP(4B9)-CEA-CD28(SA)1+1+2,三特异性二价抗CD28、单价抗FAP和单价抗CEA huIgG1 PG-LALA构建体。FAP结合剂的VH和VL结构域融合至Fc片段的相应链的C末端(FAP的VH结构域:杵链;FAP的VL结构域:臼链)。抗CEA Fab片段VH融合至具有CH1/CκCrossFab交换的FAP VH的C末端(图1K)。该分子包含SEQ ID NO:65、SEQ ID NO:107、SEQ IDNO:108和SEQ ID NO:109的氨基酸序列(P1AE0487)。
分子Z:FAP(4B9)-CEA-CD28(SA)1+1+2,三特异性二价抗CD28、单价抗FAP和单价抗CEA huIgG1 PG-LALA构建体。FAP和CEA结合剂的VH和VL结构域融合至Fc片段的相应链的C末端(FAP和CEA的VH结构域:杵链;FAP和CEA的VL结构域:臼链)(图1L)。该分子包含SEQ IDNO:62、SEQ ID NO:110和SEQ ID NO:111的氨基酸序列(P1AE0486)。
1.2靶向CD28和成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)的双特异性或三特异性抗体的生产
上述分子的表达由嵌合MPSV启动子或CMV启动子驱动。聚腺苷酸化由位于CDS的3'末端的合成polyA信号序列驱动。此外,每个载体均包含用于常染色体复制的EBV OriP序列。
为了产生构建体C至W,使用聚乙烯亚胺作为转染试剂,将在悬浮液中生长的HEK293-EBNA细胞与相应的表达载体共转染。通过瞬时转染HEK293 EBNA细胞,形成抗体和双特异性抗体。离心细胞,并将培养基替换为预热的CD CHO培养基。将表达载体在CD CHO培养基中混合,加入PEI,将溶液涡旋混合,并且在室温孵育10分钟。然后,将细胞与DNA/PEI溶液混合,将其转移至摇瓶中,并且置于培养箱中,在5%CO2的气氛下于37℃孵育3小时。孵育后,添加带有补充剂的Excell培养基((Mammalian Cell Cultures for BiologicsManufacturing,编辑:Weichang Zhou,Anne Kantardjieff)。转染后一天添加补充剂(Feed)(Mammalian Cell Cultures for Biologics Manufacturing,编辑:WeichangZhou,Anne Kantardjieff)。7天后,通过离心和随后的过滤(0.2μm过滤器)收获细胞上清液,并且使用标准方法纯化。
构建体A、B、X和Y由Evitria使用其专有的载体系统通过常规的(基于非PCR)克隆技术并且使用悬浮液适应的CHO K1细胞(最初接收自ATCC,并且适于在Evitria的悬浮液培养物中进行无血清生长)制备。在生产过程中,Evitria使用了其专有的无动物成分和无血清的培养基(eviGrow和eviMake2)及其专有的转染试剂(eviFect)。通过离心和随后的过滤(0.2μm过滤器)收获上清液,并且使用标准方法纯化。
1.3靶向CD28和成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)的双特异性或三特异性抗体的纯化
参照标准方案从过滤的细胞培养物上清液中纯化蛋白质。简而言之,使用蛋白A通过亲和色谱法从细胞培养上清液中纯化出含Fc的蛋白质。在pH 3.0下洗脱,然后立即中和样品。浓缩并凝集蛋白质,然后利用尺寸排阻色谱法在20mM组氨酸、140mM氯化钠(pH 6.0)中将聚集蛋白质与单体蛋白质分离。
1.4靶向CD28和成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)的双特异性或三特异性抗体的分析数据
通过使用根据Pace等人,Protein Science,1995,4,2411-1423的基于氨基酸序列计算的质量消光系数测量280nm处的光密度(OD),来确定纯化的构建体的蛋白质浓度。在存在和不存在还原剂的情况下,使用LabChipGXII(Perkin Elmer),通过CE-SDS分析蛋白质的纯度和分子量。利用HPLC色谱法在25℃测定聚集体含量,该系统使用分析型尺寸排阻色谱柱(TSKgel G3000 SW XL或UP-SW3000),并且在运行缓冲液(分别为25mM K2HPO4、125mMNaCl、200mM L-精氨酸盐酸盐(pH 6.7)或200mM KH2PO4、250mM KCl(pH 6.2))中平衡。表1给出了所有分子的纯化参数的总结。
表1:双特异性或三特异性CD28抗原结合分子的生产和纯化总结
实例2
靶向CD28和成纤维细胞活化蛋白(FAP)或癌胚抗原(CEA)的双特异性或三特异性抗体的双特异性抗体的结合和动力学分析
2.1靶向CD28和成纤维细胞活化蛋白(FAP)的双特异性抗体与表达FAP和CD28的细胞的结合
使用表达人成纤维细胞活化蛋白(huFAP)的3T3-huFAP细胞(克隆体19)来测试双特异性FAP-CD28分子的结合。该细胞系是通过用表达载体pETR4921在1.5μg/mL嘌呤霉素选择下转染小鼠胚胎成纤维细胞NIH/3T3细胞系(ATCC CRL-1658)以表达huFAP而生成的。用表达人CD28的CHO细胞(亲代细胞系CHO-k1 ATCC#CCL-61,经修饰以稳定地过表达人CD28)测试与人CD28的结合。
为了评估结合,收获细胞,计数,检查存活力,并以2.5E5/ml重新悬浮在FACS缓冲液(eBioscience,目录号00-4222-26)中。将5x104细胞在圆底96孔板上于4℃与递增浓度的靶向FAP的CD28构建体(1pM至100nM)一起孵育2小时。然后,将细胞用冷FACS缓冲液洗涤三次,并在4℃与PE偶联的山羊抗人PE(Jackson ImmunoReserach,目录号109-116-098)再孵育60分钟,再用冷FACS缓冲液洗涤一次,离心并重悬于100μl FACS缓冲液中。为了监测构建体和细胞之间的非特异性结合相互作用,包括抗DP47 IgG作为阴性对照。用FACS Fortessa(BD,软件FACS Diva)通过流式细胞术评估结合。使用GraphPadPrism6获得结合曲线。
FAP-CD28分子能够以浓度依赖性方式与细胞上的人FAP以及人CD28结合(某些示例如图2B和2C所示)。如预期的那样,未检测到抗DP47 IgG的结合,表明结合的检测归因于各个靶向部分的特异性CD28和FAP结合。
2.2靶向CD28和CEA的双特异性或三特异性抗体的动力学分析
使用ProteOn XPR36仪器(Biorad),使用通过中性抗生物素蛋白捕获而固定在NLC芯片上的生物素化的huCD28-Fc抗原和生物素化的Hu N(A2-B2)A-avi-His,通过SPR在25℃测量包含抗CEA(Medi-565)和抗CD28的双特异性或三特异性抗体的两个结合部分的亲和力(KD)。
为了生成含有CEA(Medi-565)表位的基于CEACAM5的抗原,生成了由两个CEACAM1结构域和两个CEACAM5 Ig结构域组成的嵌合蛋白。基于CEACAM1的序列,CEACAM1的第二和第三结构域被替换为CEACAM5结构域A2和B2。融合C末端的avi标签和His标签以进行位点特异性生物素化和纯化。所得蛋白质命名为Hu N(A2-B2)A-avi-His(SEQ ID NO:161)。
重组抗原(配体)的固定化:抗原用PBST(10mM磷酸盐、150mM氯化钠pH 7.4、0.005%吐温20)稀释至10μg/ml,然后在不同的接触时间以30μl/min的速度注射,以在垂直取向上实现约400、800和1600响应单位(RU)的固定化水平。分析物的注射:对于一次性注射的动力学测量,将注射方向更改为水平取向,以50μl/min沿着独立的通道1至5同步注射两倍稀释系列的经纯化双特异性靶向CEA的抗CD28双特异性抗体(浓度在50nM和3.125nM之间的范围内变动),缔合时间为150s,解离时间为450s。沿第六个通道注射缓冲液(PBST),以提供“在线(in-line)”空白供参考。通过同步拟合缔合和解离传感图,在ProteOn Managerv3.1软件中使用简单的一对一Langmuir结合模型来计算缔合速率常数(kon)和解离速率常数(koff)。平衡解离常数(KD)计算为比率koff/kon。包含一个抗CD28抗原结合结构域和一个抗CEA抗原结合结构域的双特异性抗体(分子M)的KD计算值与相应的单特异性构建体的测量值一致。动力学和热力学数据总结在下表2中。
表2:CEA-CD28(SA)1+1(分子M)的动力学和热力学分析
结合部分 | k<sub>on</sub>(1/(s*M) | k<sub>off</sub>(1/s) | K<sub>D</sub>(nM) |
抗CEA(Medi-565) | 4.13exp5 | 1.2exp-4 | 0.29 |
抗CD28(TGN1412) | 3.13exp5 | 3.76exp-4 | 1.2 |
实例3
没有热点并且亲和力降低的CD28(SA)变体的生成和表征
3.1未配对的半胱氨酸残基、色氨酸残基、脱酰胺位点的去除和亲和力降低的CD28(SA)变体的生成
作为我们详细的结合剂表征的一部分,对CD28(SA)可变结构域序列进行了计算分析。该分析表明VH的CDR2区中(位置50,Kabat编号)未配对的半胱氨酸,VH的CDR3中(位置100a,Kabat编号)和VL的CDR1(位置32,Kabat编号)中的色氨酸残基,以及VH的CDR2中的潜在的天冬酰胺脱酰胺作用位点(位置56,Kabat编号)。虽然色氨酸的氧化是一个相当缓慢的过程并且可以通过添加还原性化合物来防止,但是在抗体可变域中未配对的半胱氨酸的存在可能至关重要。游离半胱氨酸具有反应性,并且可以与其他蛋白质或细胞或培养基成分的其他未配对半胱氨酸形成稳定的键。结果,这可能导致具有未知修饰的异质且不稳定的产品,这些未知修饰可能具有免疫原性,因此可能给患者带来风险。此外,天冬酰胺的脱酰胺作用以及所形成的异天冬氨酸和琥珀酰亚胺可以影响体外稳定性和体内生物学功能。亲代鼠结合剂5.11A的晶体结构分析表明,C50不参与与人CD28的结合,因此可以被替换为类似的氨基酸诸如丝氨酸而不影响对CD28的亲和力(表5,变体29)。然而,色氨酸残基以及位置50处的天冬酰胺均靠近或参与结合界面,因此被替换为相似的氨基酸可导致结合亲和力降低。在这一实例中,由于以下原因,我们特别旨在降低CD28(SA)对人CD28的亲和力:CD28(SA)的亲和力在1-2nM范围内,结合半衰期约为32分钟。当静脉内注射到患者体内时,这种强大的亲和力可导致含有大量表达CD28的细胞的组织诸如血液和淋巴组织发生吸收效应。结果,可以降低经由靶向组分FAP和/或CEA对化合物的位点特异性靶向,并且可以减少构建体的功效。为了使这种效应最小化,生成了几种VH和VL变体,以将亲和力降低到不同程度(图3A和3C)。除了前面提到的代表潜在稳定性热点的位置外,直接或间接参与与人CD28结合的其他残基也被替换为原始的鼠种系氨基酸或替换为相似的氨基酸。此外,CD28(SA)VL和VH的CDR也都移植到了曲妥珠单抗的各自构架序列中(图3B和3D)。然后将VH和VL变体的几种组合表示为单价单臂抗CD28 IgG样构建体,并通过SPR表征结合。
3.2通过SPR分析经还原的单臂抗CD28变体的解离速率常数(koff)
为了在第一步中表征抗CD28结合剂变体,所有结合剂均表示为单价单臂IgG样构建体(图4A)。选择这一格式是为了在1:1模型中表征与CD28的结合。转染入HEK细胞中5天后,收集上清液并确定被表达的构建体的效价。
使用ProteOn XPR36仪器(Biorad),使用通过中性抗生物素蛋白捕获而固定在NLC芯片上的经生物素化huCD28-Fc抗原,在25℃通过表面等离振子共振(SPR)测定抗CD28结合剂变体的解离速率。对于重组抗原(配体)的固定化,将huCD28-Fc用PBST(含吐温20的磷酸盐缓冲盐水,由10mM磷酸盐、150mM氯化钠pH 7.4、0.005%吐温20组成)稀释至100nM至500nM范围的浓度,然后以不同的接触时间以25μl/min的速度注射。这导致垂直取向的固定水平在1000到3000响应单位(RU)之间。
对于一次性注射的动力学测量,将注射方向更改为水平方向。基于产生的上清液的效价,将单价单臂IgG用PBST稀释以获得100nM至6.25nM的两倍稀释系列。沿着独立的通道1至5以50μl/min的速度同步执行注射,缔合时间为120s,解离时间为300s。沿第六个通道注射缓冲液(PBST),以提供“在线(in-line)”空白供参考。由于结合相互作用是用来自上清液中的未经纯化和生化表征的单价单臂IgG测量的,因此仅将蛋白质:蛋白质相互作用的解离速率用于进一步的结论。通过拟合解离传感图,在ProteOn Manager v3.1软件中使用简单的一对一Langmuir结合模型来计算解离速率。所有克隆体的解离速率常数(koff)值总结在表2中。对所产生的变体进行的比较表明,与亲代序列相比,koff值降低了多达30倍。
表2:具有解离速率常数(koff)值的所有被表达的单价抗CD28变体的总结
3.3双特异性靶向FAP的抗CD28亲和力变体的制备和动力学分析
基于解离速率分析和对表达CD28的细胞的结合研究,选择了具有不同结合强度的抗CD28 VH和VL变体的几种组合,并表达为双特异性靶向FAP的huIgG1 PG-LALA CrossFab分子(关于多个SEQ ID NO:的组合,参见表3)。纯化得到的1+1格式的构建体(图1C),并执行生化分析(表4)。
表3:所有被表达的1+1双特异性靶向FAP的抗CD28变体的总结
表4:靶向FAP的抗CD28变体的生产和纯化总结
使用ProteOn XPR36仪器(Biorad),使用通过中性抗生物素蛋白捕获而将固定在NLC芯片上的经生物素化的huCD28-Fc抗原,在25℃通过SPR测量产生的双特异性抗原结合分子对CD28的亲和力(KD)。重组抗原(配体)的固定化:抗原用PBST(10mM磷酸盐、150mM氯化钠pH 7.4、0.005%吐温20)稀释至10μg/ml,然后在不同的接触时间以30μl/min的速度注射,以在垂直取向上实现约200、400或800响应单位(RU)的固定化水平。分析物的注射:对于一次性注射的动力学测量,将注射方向更改为水平取向,以50μl/min沿着独立的通道1至5同步注射两倍稀释系列的经纯化双特异性靶向FAP的抗CD28亲和力变体(浓度在50nM和3.125nM之间的范围内变动),缔合时间为150s,解离时间为450s。沿第六个通道注射缓冲液(PBST),以提供“在线(in-line)”空白供参考。通过同步拟合缔合和解离传感图,在ProteOnManager v3.1软件中使用简单的一对一Langmuir结合模型来计算缔合速率常数(kon)和解离速率常数(koff)。平衡解离常数(KD)计算为比率koff/kon。所分析的克隆体表明KD值范围很广(介于1nM到25nM之间)。动力学和热力学数据总结于表5中。
表5:被表达的靶向FAP的抗CD28变体的动力学和热力学分析
双特异性分子 | k<sub>on</sub>(1/(s*M) | k<sub>off</sub>(1/s) | K<sub>D</sub>(nM) |
亲代 | 3.79exp5 | 3.6exp-4 | 1 |
FAP(4B9)-CD28(CD28(SA)_变体8)1+1 | 2.19exp5 | 5.21exp-3 | 23.8 |
FAP(4B9)-CD28(CD28(SA)_变体11)1+1 | 2.3exp5 | 2.87exp-3 | 12.5 |
FAP(4B9)-CD28(CD28(SA)_变体12)1+1 | 2.6 1exp5 | 2.67exp-4 | 1 |
FAP(4B9)-CD28(CD28(SA)_变体15)1+1 | 2.59exp5 | 1.84exp-3 | 7.1 |
FAP(4B9)-CD28(CD28(SA)_变体16)1+1 | 1.87exp5 | 9.94exp-4 | 5.3 |
FAP(4B9)-CD28(CD28(SA)_变体17)1+1 | 3.38exp5 | 1.25exp-3 | 3.7 |
FAP(4B9)-CD28(CD28(SA)_变体19)1+1 | 2.8exp5 | 3.04exp-4 | 1.1 |
FAP(4B9)-CD28(CD28(SA)_变体23)1+1 | 2.11exp5 | 3.42exp-3 | 16.3 |
FAP(4B9)-CD28(CD28(SA)_变体25)1+1 | 2.38exp5 | 3.96exp-4 | 1.7 |
FAP(4B9)-CD28(CD28(SA)_变体27)1+1 | 2.27exp5 | 1.21exp-3 | 5.4 |
FAP(4B9)-CD28(CD28(SA)_变体29)1+1 | 2.72exp5 | 3.07exp-4 | 1.1 |
实例4
单价CD28激动性IgG和靶向FAP的CD28激动性抗体与表达CD28的细胞的结合
用表达人CD28的CHO细胞(亲代细胞系CHO-k1 ATCC#CCL-61,经修饰以稳定地过表达人CD28)测试与人CD28的结合。为了评估结合,收获细胞,计数,检查存活力,并以2.5x105/ml重新悬浮在FACS缓冲液(eBioscience,目录号00-4222-26)中。将5x104细胞在圆底96孔板上于4℃与递增浓度的CD28结合剂(1pM至100nM)一起孵育2小时。然后,将细胞用冷FACS缓冲液洗涤三次,并在4℃与PE偶联的山羊抗人PE(Jackson ImmunoReserach,目录号109-116-098)再孵育60分钟,再用冷FACS缓冲液洗涤一次,离心并重悬于100ul FACS缓冲液中。为了监测构建体和细胞之间的非特异性结合相互作用,包括抗DP47IgG作为阴性对照。用FACS Fortessa(BD,软件FACS Diva)通过流式细胞术评估结合。使用GraphPadPrism6获得结合曲线。
单价单臂IgG样CD28变体构建体显示结合差异,如从图4A至4C可见。此外,确定了1+1格式的双特异性靶向FAP的抗CD28抗体与表达人CD28的CHO细胞的结合。具有所选CD28变体的不同1+1构建体的KD值显示在下表6或图4D和4E的对应图中。
表6:靶向FAP的抗CD28
1+1构建体与表达人CD28的CHO细胞的结合
结合剂 | TAPIR | K<sub>D</sub>(nM) |
TGN1412 | P1AD4492 | 1 |
变体8 | P1AE3131 | 23.8 |
变体11 | P1AE3132 | 12.5 |
变体12 | P1AE3133 | 1 |
变体15 | P1AE3134 | 7.1 |
变体16 | P1AE3135 | 5.3 |
变体17 | P1AE3136 | 3.7 |
变体19 | P1AE3137 | 1.1 |
变体23 | P1AE3138 | 16.3 |
变体25 | P1AE3139 | 1.7 |
变体27 | P1AE3140 | 5.4 |
变体29 | P1AE3141 | 1.1 |
实例5
将CD28和成纤维细胞活化蛋白(FAP)靶向表达FAP和CD28的细胞的双特异性抗体的体外功能性表征
用原代人PBMC执行了几种体外测定,以评估CD28(SA)和双特异性靶向FAP的CD28抗原结合分子在存在和不存在由T细胞双特异性(TCB)抗体提供的TCR信号的情况下的活性。通过流式细胞术、细胞因子ELISA和活细胞成像确定的T细胞增殖、细胞因子分泌和肿瘤细胞杀死均作为读数获得。
2.在原代人PBMC共培养测定法中评估在不存在TCR信号的情况下双特异性靶向FAP的CD28分子的功能性,其中通过与T细胞上的人CD28和表达在3T3-huFAP细胞(亲代细胞系ATCC#CCL-92,经修饰以稳定地过表达人FAP)或表达MCSP和FAP的MV3黑色素瘤细胞上的人FAP同步结合而将双特异性靶向FAP的CD28分子交联。
3.如上所述,评估了在存在TCR信号的情况下双特异性靶向FAP的CD28分子的功能性,另外还在存在TCB分子的情况下,通过与T细胞上的CD3以及或者MKN45胃癌细胞上的人CEA(DSMZ#ACC 409)或者表达在MV3黑色素瘤细胞上的MCSP同步结合而交联。
PBMC分离
外周血单核细胞(PBMC)是通过密度梯度离心法从获自血沉棕黄层的经肝素化血液的富集淋巴细胞制备物(Blutspende Zürich)中制备的。将25ml血液(在PBS中以1:2稀释)铺展在15ml淋巴缓冲液(STEMCELL Technologies,目录号07851)上,并在室温以845xg离心25分钟,不加制动。含PBMC的中间相用10ml移液管收集在50ml试管中。用PBS洗涤细胞,并以611xg离心5分钟。弃去上清液,将沉淀重悬于50ml PBS中,并以304xg离心5分钟。重复洗涤步骤,以171xg离心。将细胞重悬于RPMI 1640Glutamax(含有5%人血清、丙酮酸钠、NEAA、50μM 2-巯基乙醇、青霉素/链霉素)中,并根据各自的测定方案进行进一步的功能分析。
PBMC的高密度预培养和CD28超激动剂CD28(SA)的T细胞活化的体外评估
为了恢复人T细胞对TGN1412介导的CD28超激动性的响应性,在评估CD28超激动性抗体的作用之前,将PBMC以高密度(HD)预培养(等人,2011)。简而言之,在完全培养基(RPMI 1640Glutamax、5%人血清、丙酮酸钠、NEAA、50uM 2-巯基乙醇、青霉素/链霉素)中将PBMC调整至1E7细胞/ml,并以1.5ml/孔在24孔板中在37℃、5%CO2下培养48小时。然后重新收获细胞,在完全培养基中洗涤,以550xg离心5分钟,并调节至进行功能表征所需的希望细胞密度。为了评估T细胞增殖,用CFSE标记PBMC,并在刺激5天后测量CFSE稀释度作为T细胞增殖的指标。简而言之,将细胞在PBS中调节至2×107/ml,并用2.5μM CFSE增殖染料(LifeTechnologies,目录号65-0850-84)在37℃、5%CO2下标记6分钟。将细胞在完全培养基中洗涤一次,然后在PBS中进行2个洗涤步骤。对于使用TGN1412刺激,在完全培养基中将PBMC调整为2x106/ml,并将1x105细胞分配到平底96孔板的每个孔中,并用递增浓度的TGN1412(0.0002nM至10nM,三次平行实验)刺激。通过流式细胞术评估CFSE稀释度。简而言之,将细胞以550xg离心5分钟,然后用PBS洗涤。通过流式细胞术评估CFSE稀释度。简而言之,将细胞以550xg离心5分钟,然后用PBS洗涤。根据供应商的指示,对CD8(BV711抗人CD8a,BioLegend#301044)、CD4(PE-Cy7抗人CD4,BioLegend#344612)进行表面染色。然后将细胞用150μl/孔PBS洗涤两次,重悬于200μl/孔的FACS缓冲液中,并使用BD FACS Fortessa进行分析。在活化后第5天,通过细胞因子ELISA分析(huTNFα,DuoSet#DY210-05和huIFNγ,DuoSet#DY285-05)或细胞因子多重分析(人细胞因子17-plex测定,Bio-Rad#M5000031YV)从培养上清液测量细胞因子的分泌。
CD28(SA)的超激动作用需要FcγRIIb交联
PBMC的高密度预培养恢复了CD28(SA)超激动作用
为了了解CD28(SA)的作用机制,我们验证了PBMC的高密度(HD)预培养作为先前描述的方案,以恢复源自PBMC的T细胞对TGN1412介导的CD28超激动作用的能力(Romer etal.,2011)。如图5A和5B所示,CD28(SA)IgG4(P1AE1975)仅在经历HD预培养的PBMC中在刺激后5天以浓度依赖的方式诱导PBMC T细胞增殖(图5A)和细胞因子生成(图5B),而在新鲜的PBMC中仍然无响应。我们得出的结论是,先前公开的在体外恢复T细胞对CD28(SA)的响应性的方案可以在我们手中再现(Romer et al.,2011)。
CD28(SA)超激动活性需要经由FcγRIIb进行的交联-FcγRIIb的阻断消除了CD28
(SA)功能性
先前公开的文献表明,TGN1412可能依赖于FcγRIIb交联。为了了解PBMC的HD预培养与CD28(SA)功能性的Fc依赖性之间的联系,在HD预培养之前和之后,通过流式细胞术评估PBMC上FcγRIIb的表达水平。如图5C所示,新鲜的PBMC单核细胞中不存在FcγRIIb表达,而在HD预培养2天后,96.8%的单核细胞表达了FcγRIIb。在5天后在培养物中测得,当使用CD28(SA)刺激后,抗体介导的FcγRIIb的阻断在后续T细胞增殖测定中完全消除了T细胞增殖(图5D)。在替代方法中,携带P329G-LALA突变的CD28(SA)的Fc沉默变体(CD28(SA)IgG1PG-LALA:P1AD9289)未展示超激动性功能(图6A)。这些数据证实,CD28(SA)介导的CD28超激动作用依赖于通过FcγRIIb进行的交联。
向Fc沉默的CD28(SA)添加靶向FAP的肿瘤靶向部分恢复了超激动作用,然后,超激
动作用取决于肿瘤靶的存在
假定通过CD28(SA)产生的CD28超激动作用依赖于FcγRIIb交联,我们假设FcR依赖性可以通过引入以下项而重新定向到肿瘤:(i)沉默Fc的P329G-LALA突变和(ii)与表面表达的肿瘤抗原交联的靶向部分。为了测试这一假设,将FAP靶向部分作为C末端融合体添加到Fc沉默的CD28(SA)(FAP-CD28(SA)1+2:P1AD9011)。由于这一方法不需要FcR交联,因此PBMC无需进行HD预培养。取而代之的是,将新鲜的PBMC与3T3-huFAP或3T3-WT在存在递增浓度的FAP-CD28(P1AD9011)的情况下共培养5天,并通过以流式细胞术测得的CFSE稀释度来评估T细胞增殖。如图6B所示,FAP结合部分的引入使T细胞仅在存在FAP的情况下才能增殖。我们得出的结论是,超激动作用可以通过沉默Fc和添加肿瘤靶向部分而选择性地靶向肿瘤抗原。
在不存在和存在TCB信号的情况下,通过双特异性靶向FAP的CD28抗原结合分子在
体外评估T细胞增殖和细胞因子分泌
Pan T细胞用作效应细胞,并根据制造商的说明使用Pan T细胞分离试剂盒(Miltenyi Biotec)通过MACS从PBMC中分离出来。
为了在不存在TCB的情况下通过双特异性FAP-CD28抗原结合分子测量T细胞活化,将CFSE标记的pan T细胞与前一天接种在平底96孔板中的3x104/孔的3T3-huFAP或缺乏FAP表达的亲代3T3细胞(3T3-WT)共培养。以递增的浓度(0.0002nM至10nM,三次平行实验)添加双特异性FAP-CD28抗原结合分子。
为了在存在TCB信号的情况下测量T细胞增殖,将CFSE标记的pan T细胞与前一天接种在平底96孔板中的每孔3x104个表达FAP和MCSP的MV3细胞一起孵育,以递增浓度的双特异性FAP-CD28抗原结合分子(0.0002nM至10nM,三次平行实验)和固定浓度的MCSP-TCB(5pM,P1AD2189)进行。作为对照,包括仅包含TCB的孔。
通过流式细胞术评估CFSE稀释度,并且在活化后第5天,通过细胞因子ELISA分析(huTNFα,DuoSet#DY210-05和huIFNγ,DuoSet#DY285-05)或细胞因子多重分析(人细胞因子17-plex测定,Bio-Rad#M5000031YV)从培养上清液测量细胞因子的分泌。
常规CD28激动性抗体(克隆体9.3)在靶向肿瘤的双特异性格式下不具有超激动性
文献中已经报道了两种类型的CD28激动性抗体:超激动性CD28抗体(诸如TGN1412)能够自主活化T细胞,而无需TCR提供的另外的信号。这些抗体被称为超激动剂,因为它们超越了天然CD28激动性配体CD80和CD86的功能性,后两者严格依赖于TCR信号的存在来增强T细胞功能。与超激动性抗体(诸如TGN1412)相比,常规激动性抗体(诸如克隆体mab 9.3)不能自主活化T细胞,而是就像天然CD28配体一样需要,另外的TCR信号来增强T细胞活性。为了更详细地评估将CD28激动剂靶向肿瘤抗原的作用,我们生成了更多的FAP-CD28分子:(i)具有2个CD28结合部分(TGN1412)和2个FAP结合部分=2+2SA格式的超激动性(SA)分子(P1AD4493),(ii)具有2个CD28结合部分(克隆体9.3)和1或2个FAP结合部分的常规激动剂(CA),分别为:2+2CA(P1AD8968)、1+2CA(P1AD8962)。将新鲜的PBMC与3T3-huFAP或3T3-WT在存在递增浓度的靶向FAP的分子的情况下共培养5天,并通过以流式细胞术测得的CFSE稀释度来评估T细胞增殖。如图7A至7D所示,仅超激动性结合剂能够活化T细胞。此外,通过所描述的超激动性构建体进行的T细胞活化严格依赖于FAP的存在(图7B),如在不存在FAP的情况下不存在T细胞活化所证实的(图7D)。与这些数据一致,也仅观察到具有超激动性CD28(SA)抗体的构建体的细胞因子分泌,而没有观察到常规激动性9.3抗体的细胞因子分泌(图7E)。我们得出的结论是,只有超激动性CD28抗体会以双特异性靶向肿瘤的抗体格式引发自主性T细胞活化,而具有常规9.3结合剂的相同格式并非超激动性的。
实例6
对通过靶向肿瘤的CD28分子在不存在或未存在TCB的情况下进行的肿瘤细胞杀死的体外评估
为了评估双特异性FAP-CD28或CEA-CD28抗原结合分子实现肿瘤细胞杀死或支持TCB介导的肿瘤细胞杀死的能力,将纯化的pan T细胞用作效应细胞,并将表达RFP的MV3细胞和MKN45细胞分别用作肿瘤靶。
为了评估MV3肿瘤细胞的杀死,在存在单独的或与10nM双特异性FAP-CD28抗原结合分子结合使用的5pM MCSP-TCB(P1AD2189)的情况下,在平底96孔板(E:T 20:1)的每个孔中将前一天接种的5000MV3靶细胞与1x105 pan T细胞共培养。为了评估MV3肿瘤细胞的杀死,在存在2nM FAP-CD28的情况下,在平底96孔板(E:T 20:1)的每个孔中将前一天接种的5000MV3靶细胞与1x105 pan T细胞共培养。为了评估MKN45肿瘤细胞的杀死,在存在2nMCEA-CD28的情况下,在平底96孔板的每个孔中将前一天接种的5000MKN45与1x105 pan T细胞共培养。使用IncuCyte活细胞成像系统(Essen Biosciences)在90小时的进程中监控靶细胞的杀死情况,每3小时每孔捕获4张图像。随着时间的推移,每个图像的RFP+对象计数(通过IncuCyte ZOOM软件(Essen Biosciences)评估)可作为靶细胞死亡的指标。通过监测在存在效应T细胞的情况下随时间推移的靶细胞计数,将抗体介导的靶细胞杀死与自发性靶细胞死亡区分开(=基线对照)。杀死计算为100-x,x是相对于基线对照的靶百分比。使用学生t检验进行统计分析,比较随时间推移的杀死百分比的曲线下面积(AUC)。
2+1格式的FAP-CD28诱导靶细胞杀死,但仅具有超激动性CD28结合剂者有此效应,
而具有常规CD28激动性结合剂者无此效应
评估了FAP-CD28分子诱导肿瘤细胞杀死的能力。如图8A至8D所示,在存在FAP-CD28的情况下,将源自PBMC的T细胞与表达FAP的MV3黑色素瘤细胞共培养90小时,使得MV3细胞的杀死唯一性地由1+2格式的FAP CD28(SA)(P1AD9011)导致,并且与靶向FAP的TCB(P1AD4645)诱导的杀死作用相当。使用2+2格式的FAP-CD28(SA)(P1AD4493)和具有常规CD28激动性9.3抗体(P1AD8968和P1AD8962)的FAP-CD28均未观察到杀死作用。我们得出的结论是,除了T细胞增殖和细胞因子分泌外,具有超激动性结合剂的1+2格式的FAP-CD28还可以引起靶细胞杀死,与TCB相当。
1+2和2+2格式的CEA-CD28诱导靶细胞杀死,但仅具有超激动性抗体者有此效应,
而具有常规CD28激动性抗体者无此效应
在另一替代性方法中,我们使用2+2SA(P1AE1195)、1+2SA(P1AE1194)、2+2CA(P1AE1193)和2+1CA(P1AE1192)格式的靶向CEA的CD28激动性分子来评估它们诱导靶细胞杀死的能力。在存在前述格式的CEA-CD28的情况下,将PBMC T细胞与表达CEA的MKN45细胞共培养90小时。包含超激动性CD28结合剂的两种格式都能够诱导表达CEA的MKN45细胞被杀死(图9A和9B)。我们推测,FAP-CD28(SA)2+2和CEA-CD28(SA)2+2杀死其各自靶细胞的能力之间的差异在于MKN45细胞与MV3细胞中靶标表达水平的差异。准确地,内部数据证实,MV3细胞的FAP表达水平比MKN45细胞的CEA表达水平低10倍。因此,在MV3细胞中,肿瘤靶结合位点可能是限制性的,并且杀死MV3细胞需要FAP与CD28相比的有效占位,这在1+2格式(即1个FAP结合位点使得2个CD28结合位点交联)中比在2+2格式(即2个CD28结合位点的交联需要2个FAP结合位点)中有利。
通过TGN1412结合剂产生的CD28超激动作用依赖于CD28结合剂的多重价-单价结
合剂不是超激动性的
为了进一步研究CD28超激动作用的属性,我们评估了单价CD28TGN1412结合剂是否以靶向肿瘤的双特异性形式显示超激动性行为。将PBMC T细胞与3T3-huFAP细胞共培养,并与递增浓度的具有CD28双价的FAP-CD28 1+2SA(P1AD9011)和具有CD28单价的FAP-CD281+1SA(P1AD4492)一起孵育。如图10A所示,与CD28二价构建体(P1AD9011)相反,具有单价CD28结合的FAP-CD28(P1AD4492)不能诱导T细胞增殖。一致地,仅在CD28二价的情况下观察到T细胞活化标志物CD69和CD25的上调(分别为图10B和10C)。总之,TGN1412介导的超激动作用不仅依赖于通过Fc受体进行的交联,而且要求CD28结合剂具有多重价。
总之,可以确认,可以通过沉默Fc和引入能够与肿瘤相关抗原特异性结合的抗原结合结构域将CD28超激动性作用特异性地靶向肿瘤抗原。此外,靶向肿瘤的双特异性抗体仅当它们包含基于CD28(SA)的结合剂时才是超激动性的,而当它们包含常规激动性结合剂(克隆体9.3)时不是超激动性的。此外,超激动性要求CD28(SA)结合剂具有多重价,而双特异性构建体中的单价CD28(SA)结合则消除了超激动性T细胞活化。
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Engelhardt,J.J.,Sullivan,T.J.,and Allison,J.P.(2006).CTLA-4overexpression inhibits T cell responses through a CD28-B7-dependentmechanism.J Immunol 177,1052-1061.
Esensten,J.H.,Helou,Y.A.,Chopra,G.,Weiss,A.,and Bluestone,J.A.(2016).CD28 Costimulation:From Mechanism to Therapy.Immunity 44,973-988.
Fraser,J.D.,Irving,B.A.,Crabtree,G.R.,and Weiss,A.(1991).Regulationof interleukin-2 gene enhancer activity by the T cell accessory moleculeCD28.Science 251,313-316.
Hui,E.,Cheung,J.,Zhu,J.,Su,X.,Taylor,M.J.,Wallweber,H.A.,Sasmal,D.K.,Huang,J.,Kim,J.M.,Mellman,I.,and Vale,R.D.(2017).T cell costimulatoryreceptor CD28 is a primary target for PD-1-mediated inhibition.Science 355,1428-1433.
Hunig,T.(2012).The storm has cleared:lessons from theCD28superagonist TGN1412 trial.Nat Rev Immunol 12,317-318.
June,C.H.,Ledbetter,J.A.,Gillespie,M.M.,Lindsten,T.,and Thompson,C.B.(1987)。T-cell proliferation involving the CD28 pathway is associated withcyclosporine-resistant interleukin 2 gene expression.Mol Cell Biol 7,4472-4481.
Kamphorst,A.O.,Wieland,A.,Nasti,T.,Yang,S.,Zhang,R.,Barber,D.L.,Konieczny,B.T.,Daugherty,C.Z.,Koenig,L.,Yu,K.,et al.(2017).Rescue ofexhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent.Science355,1423-1427.
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序列表
<110> 豪夫迈·罗氏有限公司(F. Hoffmann-La Roche AG)
<120> 靶向肿瘤的超激动性CD28抗原结合分子
<130> P35127-WO
<150> EP18215116.7
<151> 2018-12-21
<160> 162
<170> PatentIn 版本 3.5
<210> 1
<211> 220
<212> PRT
<213> 智人
<400> 1
Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val
1 5 10 15
Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr
20 25 30
Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser
35 40 45
Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu
50 55 60
Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser
65 70 75 80
Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr
85 90 95
Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys
100 105 110
Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
115 120 125
Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
130 135 140
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly
145 150 155 160
Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile
165 170 175
Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
180 185 190
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
195 200 205
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
210 215 220
<210> 2
<211> 760
<212> PRT
<213> 智人
<400> 2
Met Lys Thr Trp Val Lys Ile Val Phe Gly Val Ala Thr Ser Ala Val
1 5 10 15
Leu Ala Leu Leu Val Met Cys Ile Val Leu Arg Pro Ser Arg Val His
20 25 30
Asn Ser Glu Glu Asn Thr Met Arg Ala Leu Thr Leu Lys Asp Ile Leu
35 40 45
Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe Pro Asn Trp Ile Ser Gly
50 55 60
Gln Glu Tyr Leu His Gln Ser Ala Asp Asn Asn Ile Val Leu Tyr Asn
65 70 75 80
Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu Ser Asn Arg Thr Met Lys
85 90 95
Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser Pro Asp Arg Gln Phe Val
100 105 110
Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala
115 120 125
Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly Glu Phe Val Arg Gly Asn
130 135 140
Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser
145 150 155 160
Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro
165 170 175
Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn Gly Arg Glu Asn Lys Ile
180 185 190
Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu Ala Thr
195 200 205
Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly Lys Phe Leu Ala Tyr Ala
210 215 220
Glu Phe Asn Asp Thr Asp Ile Pro Val Ile Ala Tyr Ser Tyr Tyr Gly
225 230 235 240
Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly
245 250 255
Ala Lys Asn Pro Val Val Arg Ile Phe Ile Ile Asp Thr Thr Tyr Pro
260 265 270
Ala Tyr Val Gly Pro Gln Glu Val Pro Val Pro Ala Met Ile Ala Ser
275 280 285
Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Thr Asp Glu Arg Val
290 295 300
Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu Ser Ile
305 310 315 320
Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys Thr Gln
325 330 335
Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val
340 345 350
Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe
355 360 365
Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val
370 375 380
Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Asn Ile
385 390 395 400
Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu
405 410 415
Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Ser Tyr
420 425 430
Pro Pro Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg Cys
435 440 445
Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala Leu
450 455 460
Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser Thr Leu His Asp Gly Arg
465 470 475 480
Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu Asn Lys Glu Leu Glu Asn
485 490 495
Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu Glu Ile Lys Lys Leu Glu
500 505 510
Val Asp Glu Ile Thr Leu Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe
515 520 525
Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro
530 535 540
Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile Ser Tyr
545 550 555 560
Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val Asp Gly Arg Gly
565 570 575
Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr Ala Val Tyr Arg Lys Leu
580 585 590
Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val Arg Lys Phe Ile
595 600 605
Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile Trp Gly Trp Ser
610 615 620
Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu Ala Ser Gly Thr Gly Leu
625 630 635 640
Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr
645 650 655
Ala Ser Val Tyr Thr Glu Arg Phe Met Gly Leu Pro Thr Lys Asp Asp
660 665 670
Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala Glu Tyr
675 680 685
Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp Asp Asn
690 695 700
Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val Asn Ala
705 710 715 720
Gln Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His Gly Leu
725 730 735
Ser Gly Leu Ser Thr Asn His Leu Tyr Thr His Met Thr His Phe Leu
740 745 750
Lys Gln Cys Phe Ser Leu Ser Asp
755 760
<210> 3
<211> 702
<212> PRT
<213> 智人
<400> 3
Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln
1 5 10 15
Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr
20 25 30
Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly
35 40 45
Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly
50 55 60
Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile
65 70 75 80
Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser
85 90 95
Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile
100 105 110
Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp
115 120 125
Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu
130 135 140
Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys
145 150 155 160
Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr
165 170 175
Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln
180 185 190
Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn
195 200 205
Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg
210 215 220
Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro
225 230 235 240
Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn
245 250 255
Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe
260 265 270
Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn
275 280 285
Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser
290 295 300
Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala
305 310 315 320
Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu
325 330 335
Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr
340 345 350
Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg
355 360 365
Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr
370 375 380
Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser
385 390 395 400
Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp
405 410 415
Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn
420 425 430
Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser
435 440 445
Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile
450 455 460
Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn
465 470 475 480
Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val
485 490 495
Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro
500 505 510
Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln
515 520 525
Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser
530 535 540
Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn
545 550 555 560
Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser
565 570 575
Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly
580 585 590
Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly
595 600 605
Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln
610 615 620
Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu
625 630 635 640
Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe
645 650 655
Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile
660 665 670
Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr
675 680 685
Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile
690 695 700
<210> 4
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) CDR-H1
<400> 4
Ser His Ala Met Ser
1 5
<210> 5
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) CDR-H2
<400> 5
Ala Ile Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 6
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) CDR-H3
<400> 6
Gly Trp Leu Gly Asn Phe Asp Tyr
1 5
<210> 7
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) CDR-L1
<400> 7
Arg Ala Ser Gln Ser Val Ser Arg Ser Tyr Leu Ala
1 5 10
<210> 8
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) CDR-L2
<400> 8
Gly Ala Ser Thr Arg Ala Thr
1 5
<210> 9
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) CDR-L3
<400> 9
Gln Gln Gly Gln Val Ile Pro Pro Thr
1 5
<210> 10
<211> 116
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) VH
<400> 10
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 Ser Ser His
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys Gly Trp Leu Gly Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 11
<211> 108
<212> PRT
<213> 人工序列
<220>
<223> FAP(28H1) VL
<400> 11
Glu Ile Val Leu Thr Gln Ser Pro Gly 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 Arg Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Ile Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Gln Val Ile Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 12
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) CDR-H1
<400> 12
Ser Tyr Ala Met Ser
1 5
<210> 13
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) CDR-H2
<400> 13
Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 14
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) CDR-H3
<400> 14
Gly Trp Phe Gly Gly Phe Asn Tyr
1 5
<210> 15
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) CDR-L1
<400> 15
Arg Ala Ser Gln Ser Val Ser Arg Ser Tyr Leu Ala
1 5 10
<210> 16
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) CDR-L2
<400> 16
Val Gly Ser Arg Arg Ala Thr
1 5
<210> 17
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) CDR-L3
<400> 17
Gln Gln Gly Ile Met Leu Pro Pro Thr
1 5
<210> 18
<211> 117
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VH
<400> 18
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 Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ile Gly Ser Gly Ala Ser 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 Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 19
<211> 108
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VL
<400> 19
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 20
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) CDR-H1
<400> 20
Ser Tyr Tyr Ile His
1 5
<210> 21
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) CDR-H2
<400> 21
Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 22
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) CDR-H3
<400> 22
Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val
1 5 10
<210> 23
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) CDR-L1
<400> 23
His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn
1 5 10
<210> 24
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) CDR-L2
<400> 24
Lys Ala Ser Asn Leu His Thr
1 5
<210> 25
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) CDR-L3
<400> 25
Gln Gln Gly Gln Thr Tyr Pro Tyr Thr
1 5
<210> 26
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VH
<400> 26
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 27
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VL
<400> 27
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 His Ala Ser Gln Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 28
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) CDR-H1
<400> 28
Asp Tyr Gly Val His
1 5
<210> 29
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) CDR-H2
<400> 29
Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met Ser
1 5 10 15
<210> 30
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) CDR-H3
<400> 30
Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr
1 5 10
<210> 31
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) CDR-L1
<400> 31
Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gln
1 5 10 15
<210> 32
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) CDR-L2
<400> 32
Ala Ala Ser Asn Val Glu Ser
1 5
<210> 33
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) CDR-L3
<400> 33
Gln Gln Ser Arg Lys Val Pro Tyr Thr
1 5
<210> 34
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VH
<400> 34
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 35
<211> 111
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VL
<400> 35
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 36
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> CD28 CDR-H1 共有
<400> 36
Ser Tyr Tyr Ile His
1 5
<210> 37
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> CD28 CDR-H2 共有
<220>
<221> 变体
<222> (5)..(5)
<223> Gly 或 Arg
<220>
<221> 变体
<222> (6)..(6)
<223> Asn 或 Asp
<220>
<221> 变体
<222> (7)..(7)
<223> Val 或 Gly
<220>
<221> 变体
<222> (8)..(8)
<223> Asn、Gln 或 Ala
<400> 37
Ser Ile Tyr Pro Xaa Xaa Xaa Xaa Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Asp
<210> 38
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> CD28 CDR-H3 共有
<220>
<221> 变体
<222> (5)..(5)
<223> Leu 或 Ala
<220>
<221> 变体
<222> (7)..(7)
<223> Trp、His、Tyr 或 Phe
<400> 38
Ser His Tyr Gly Xaa Asp Xaa Asn Phe Asp Val
1 5 10
<210> 39
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> CD28 CDR-L1 共有
<220>
<221> 变体
<222> (1)..(1)
<223> His 或 Arg
<220>
<221> 变体
<222> (5)..(5)
<223> Asn 或 Gly
<220>
<221> 变体
<222> (7)..(7)
<223> Tyr 或 Ser
<220>
<221> 变体
<222> (8)..(8)
<223> Val 或 Asn
<220>
<221> 变体
<222> (9)..(9)
<223> Trp、His、Phe 或 Tyr
<400> 39
Xaa Ala Ser Gln Xaa Ile Xaa Xaa Xaa Leu Asn
1 5 10
<210> 40
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> CD28 CDR-L2 共有
<220>
<221> 变体
<222> (1)..(1)
<223> Lys 或 Tyr
<220>
<221> 变体
<222> (2)..(2)
<223> Ala 或 Gly
<220>
<221> 变体
<222> (4)..(4)
<223> Asn 或 Ser
<220>
<221> 变体
<222> (6)..(6)
<223> His 或 Tyr
<220>
<221> 变体
<222> (7)..(7)
<223> Thr 或 Ser
<400> 40
Xaa Xaa Ser Xaa Leu Xaa Xaa
1 5
<210> 41
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> CD28 CDR-L3 共有
<220>
<221> 变体
<222> (3)..(3)
<223> Gly 或 Ala
<400> 41
Gln Gln Xaa Gln Thr Tyr Pro Tyr Thr
1 5
<210> 42
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 a
<400> 42
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 43
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 b
<400> 43
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp His Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 44
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 c
<400> 44
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Ala Asp His Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 45
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 d
<400> 45
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Arg Asp Gly Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Tyr Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 46
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 e
<400> 46
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 47
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 f
<400> 47
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Phe Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 48
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 g
<400> 48
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Arg Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp His Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 49
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 h
<400> 49
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Arg Asp Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp His Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 50
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 i
<400> 50
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 Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Tyr Pro Gly Asn Val Asn Thr Arg 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
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 51
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> CD28 VH 变体 j
<400> 51
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 Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Tyr Pro Gly Asn Val Ala Thr Arg 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
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 52
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 k
<400> 52
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 His Ala Ser Gln Asn Ile Tyr Val His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Ala Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 53
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 l
<400> 53
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 His Ala Ser Gln Asn Ile Tyr Val Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 54
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 m
<400> 54
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 His Ala Ser Gln Asn Ile Tyr Val Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 55
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 n
<400> 55
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 His Ala Ser Gln Gly 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 Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 56
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 o
<400> 56
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 His Ala Ser Gln Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Ser Leu His 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 57
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 p
<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 His Ala Ser Gln Gly 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 Tyr Thr Ser Ser Leu His 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 58
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 q
<400> 58
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 His Ala Ser Gln Gly Ile Ser Asn His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 59
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 r
<400> 59
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 His Ala Ser Gln Gly Ile Tyr Val Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 60
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 s
<400> 60
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 His Ala Ser Gln Gly Ile Ser Val Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 61
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> CD28 VL 变体 t
<400> 61
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 Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 62
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) 轻链
<400> 62
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 His Ala Ser Gln Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr 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
210
<210> 63
<211> 447
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) hu IgG4 重链
<400> 63
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220
Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
260 265 270
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 64
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) hu IgG1 PGLALA 重链
<400> 64
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly 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
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
<210> 65
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) hu IgG 轻链“RK”
<400> 65
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 His Ala Ser Gln Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr 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 Arg Lys 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> 66
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) hu IgG1 PGLALA Fc 杵
<400> 66
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 67
<211> 438
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VL-CH hu IgG1 PGLALA Fc 臼
<400> 67
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser
100 105 110
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
115 120 125
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
130 135 140
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
145 150 155 160
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
165 170 175
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
180 185 190
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
195 200 205
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
210 215 220
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
225 230 235 240
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
245 250 255
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
260 265 270
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
275 280 285
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
290 295 300
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
305 310 315 320
Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
325 330 335
Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
340 345 350
Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
355 360 365
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
370 375 380
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val
385 390 395 400
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
405 410 415
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
420 425 430
Ser Leu Ser Leu Ser Pro
435
<210> 68
<211> 224
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VH-Cκ
<400> 68
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 Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ile Gly Ser Gly Ala Ser 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 Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe
115 120 125
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
130 135 140
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
145 150 155 160
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
165 170 175
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
180 185 190
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
195 200 205
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 69
<211> 820
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH-VHCH Fc 杵 FAP(4B9) VH PGLALA
<400> 69
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
225 230 235 240
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
245 250 255
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Val Arg
260 265 270
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Cys Ile Tyr Pro Gly
275 280 285
Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe Lys Asp Arg Ala Thr Leu
290 295 300
Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
305 310 315 320
Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Thr Arg Ser His Tyr Gly
325 330 335
Leu Asp Trp Asn Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser
675 680 685
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
690 695 700
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
705 710 715 720
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
725 730 735
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
740 745 750
Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys
755 760 765
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
770 775 780
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
785 790 795 800
Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val
805 810 815
Thr Val Ser Ser
820
<210> 70
<211> 811
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH-VHCH Fc 臼 FAP(4B9) VL PGLALA
<400> 70
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
225 230 235 240
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
245 250 255
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Val Arg
260 265 270
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Cys Ile Tyr Pro Gly
275 280 285
Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe Lys Asp Arg Ala Thr Leu
290 295 300
Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu
305 310 315 320
Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Thr Arg Ser His Tyr Gly
325 330 335
Leu Asp Trp Asn Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser
675 680 685
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
690 695 700
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu
705 710 715 720
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser Tyr
725 730 735
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
740 745 750
Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
755 760 765
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
770 775 780
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro Pro
785 790 795 800
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
805 810
<210> 71
<211> 586
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH- Fc 杵 FAP(4B9) VH
<400> 71
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly 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 Cys 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
515 520 525
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
530 535 540
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
545 550 555 560
Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp
565 570 575
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
580 585
<210> 72
<211> 577
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH- Fc 臼 FAP(4B9) VL
<400> 72
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
465 470 475 480
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
485 490 495
Ser Val Thr Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
500 505 510
Ala Pro Arg Leu Leu Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile
515 520 525
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
530 535 540
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
545 550 555 560
Gly Ile Met Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
565 570 575
Lys
<210> 73
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH "EE"- Fc PGLALA FAP(4B9) VHCL
<400> 73
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
515 520 525
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
530 535 540
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
545 550 555 560
Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp
565 570 575
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro
580 585 590
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
595 600 605
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
610 615 620
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
625 630 635 640
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
645 650 655
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
660 665 670
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
675 680 685
Asn Arg Gly Glu Cys
690
<210> 74
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VLCH1
<400> 74
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser
100 105 110
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
115 120 125
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
130 135 140
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
145 150 155 160
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
165 170 175
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
180 185 190
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
195 200 205
Glu Pro Lys Ser Cys Asp
210
<210> 75
<211> 668
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VLCH1- FAP(4B9) VHCH1 "EE"- Fc 杵 PGLALA
<400> 75
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 His Ala Ser Gln Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr
100 105 110
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
115 120 125
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
130 135 140
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
145 150 155 160
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
165 170 175
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
180 185 190
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
195 200 205
Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
210 215 220
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
225 230 235 240
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
245 250 255
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
260 265 270
Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys
275 280 285
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
290 295 300
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
305 310 315 320
Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val
325 330 335
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
340 345 350
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
355 360 365
Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
370 375 380
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
385 390 395 400
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
405 410 415
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
420 425 430
Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
435 440 445
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
450 455 460
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
465 470 475 480
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
485 490 495
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
500 505 510
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
515 520 525
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
530 535 540
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
545 550 555 560
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
565 570 575
Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
580 585 590
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
595 600 605
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
610 615 620
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
625 630 635 640
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
645 650 655
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
660 665
<210> 76
<211> 445
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VHCH1 "EE"- Fc 臼 PGLALA
<400> 76
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 Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ile Gly Ser Gly Ala Ser 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 Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
<210> 77
<211> 227
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCL
<400> 77
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys
225
<210> 78
<211> 215
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VLCL "RK"
<400> 78
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 79
<211> 225
<212> PRT
<213> 人工序列
<220>
<223> Fc 臼 PGLALA
<400> 79
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro
225
<210> 80
<211> 363
<212> PRT
<213> 人工序列
<220>
<223> Fc 杵 -FAP(4B9) VH
<400> 80
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
245 250 255
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
260 265 270
Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly
275 280 285
Lys Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr
290 295 300
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
305 310 315 320
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
325 330 335
Thr Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr
340 345 350
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
355 360
<210> 81
<211> 697
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1 "EE"- Fc PGLALA CEA(Medi-565) VHCL
<400> 81
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Val Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr
515 520 525
Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
530 535 540
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
545 550 555 560
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr
565 570 575
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser
580 585 590
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
595 600 605
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
610 615 620
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
625 630 635 640
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
645 650 655
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
660 665 670
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
675 680 685
Thr Lys Ser Phe Asn Arg Gly Glu Cys
690 695
<210> 82
<211> 221
<212> PRT
<213> 人工序列
<220>
<223> CEA-VLCH1
<400> 82
Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala
1 5 10 15
Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly Ile Asn Val Gly Ala
20 25 30
Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr
35 40 45
Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val
50 55 60
Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile
65 70 75 80
Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Met Ile Trp His Ser Gly Ala Ser Ala Val Phe Gly Gly Gly Thr Lys
100 105 110
Leu Thr Val Leu 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
210 215 220
<210> 83
<211> 590
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1- Fc 杵 CEA VH
<400> 83
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly 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 Cys 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Val Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr
515 520 525
Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
530 535 540
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
545 550 555 560
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr
565 570 575
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
580 585 590
<210> 84
<211> 585
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1- Fc 臼 CEA VL
<400> 84
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser
465 470 475 480
Ala Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly
485 490 495
Ile Asn Val Gly Ala Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly
500 505 510
Ser Pro Pro Gln Tyr Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln
515 520 525
Gln Gly Ser Gly Val Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser
530 535 540
Ala Asn Ala Gly Ile Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu
545 550 555 560
Ala Asp Tyr Tyr Cys Met Ile Trp His Ser Gly Ala Ser Ala Val Phe
565 570 575
Gly Gly Gly Thr Lys Leu Thr Val Leu
580 585
<210> 85
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1 "EE"- Fc 臼 PGLALA HYRF
<400> 85
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
<210> 86
<211> 225
<212> PRT
<213> 人工序列
<220>
<223> Fc 杵 PGLALA
<400> 86
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro
225
<210> 87
<211> 446
<212> PRT
<213> 人工序列
<220>
<223> CEA VL-CH hu IgG1 PGLALA Fc 臼
<400> 87
Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala
1 5 10 15
Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly Ile Asn Val Gly Ala
20 25 30
Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr
35 40 45
Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val
50 55 60
Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile
65 70 75 80
Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Met Ile Trp His Ser Gly Ala Ser Ala Val Phe Gly Gly Gly Thr Lys
100 105 110
Leu Thr Val Leu 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 Ala Ala 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 Gly Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 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
435 440 445
<210> 88
<211> 228
<212> PRT
<213> 人工序列
<220>
<223> CEA VH-CL
<400> 88
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 Val Ser Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser
115 120 125
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
130 135 140
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
145 150 155 160
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
165 170 175
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
180 185 190
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
195 200 205
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
210 215 220
Arg Gly Glu Cys
225
<210> 89
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) 轻链
<400> 89
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 90
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) hu IgG1 PGLALA 重链
<400> 90
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Ala Ala 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 Gly 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
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
<210> 91
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) hu IgG1 PGLALA Fc 杵 "EE"
<400> 91
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 92
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) hu IgG 轻链 "RK"
<400> 92
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 93
<211> 820
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH-VHCH Fc 杵 FAP(4B9) VH PGLALA
<400> 93
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Gln
225 230 235 240
Ser Gly Pro Gly Leu Val Thr Pro Ser Gln Ser Leu Ser Ile Thr Cys
245 250 255
Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr Gly Val His Trp Val Arg
260 265 270
Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly
275 280 285
Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Lys Ser Ile Ser
290 295 300
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
305 310 315 320
Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Lys Gly Tyr Ser
325 330 335
Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser
675 680 685
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
690 695 700
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
705 710 715 720
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
725 730 735
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
740 745 750
Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys
755 760 765
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
770 775 780
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
785 790 795 800
Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val
805 810 815
Thr Val Ser Ser
820
<210> 94
<211> 811
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH-VHCH Fc 臼 FAP(4B9) VL PGLALA
<400> 94
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Gln
225 230 235 240
Ser Gly Pro Gly Leu Val Thr Pro Ser Gln Ser Leu Ser Ile Thr Cys
245 250 255
Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr Gly Val His Trp Val Arg
260 265 270
Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly
275 280 285
Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Lys Ser Ile Ser
290 295 300
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
305 310 315 320
Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Lys Gly Tyr Ser
325 330 335
Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
340 345 350
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
355 360 365
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
370 375 380
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
385 390 395 400
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
405 410 415
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
420 425 430
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
435 440 445
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
450 455 460
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
465 470 475 480
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
485 490 495
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
500 505 510
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
515 520 525
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
530 535 540
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
545 550 555 560
Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
565 570 575
Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
580 585 590
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
595 600 605
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
610 615 620
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
625 630 635 640
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
645 650 655
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
660 665 670
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser
675 680 685
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
690 695 700
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu
705 710 715 720
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser Tyr
725 730 735
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
740 745 750
Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
755 760 765
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
770 775 780
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro Pro
785 790 795 800
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
805 810
<210> 95
<211> 586
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH- Fc 杵 FAP(4B9) VH
<400> 95
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Ala Ala 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 Gly 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 Cys 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
515 520 525
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
530 535 540
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
545 550 555 560
Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp
565 570 575
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
580 585
<210> 96
<211> 577
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH- Fc 臼 FAP(4B9) VL
<400> 96
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
465 470 475 480
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
485 490 495
Ser Val Thr Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
500 505 510
Ala Pro Arg Leu Leu Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile
515 520 525
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
530 535 540
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
545 550 555 560
Gly Ile Met Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
565 570 575
Lys
<210> 97
<211> 693
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH "EE"- Fc PGLALA FAP(4B9) VHCL
<400> 97
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
515 520 525
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
530 535 540
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
545 550 555 560
Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp
565 570 575
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro
580 585 590
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
595 600 605
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
610 615 620
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
625 630 635 640
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
645 650 655
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
660 665 670
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
675 680 685
Asn Arg Gly Glu Cys
690
<210> 98
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VLCL "RK"
<400> 98
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 99
<211> 213
<212> PRT
<213> 人工序列
<220>
<223> FAP(4B9) VL-CH1
<400> 99
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro
85 90 95
Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser
100 105 110
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
115 120 125
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
130 135 140
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
145 150 155 160
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
165 170 175
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
180 185 190
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
195 200 205
Glu Pro Lys Ser Cys
210
<210> 100
<211> 672
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VLCH1- FAP(4B9) VHCH1 "EE"- Fc 杵 PGLALA
<400> 100
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 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 Gly Gly Gly Gly Ser Gly Gly
210 215 220
Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
225 230 235 240
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
245 250 255
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
260 265 270
Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala
275 280 285
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
290 295 300
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
305 310 315 320
Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln
325 330 335
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
340 345 350
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
355 360 365
Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
370 375 380
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
385 390 395 400
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
405 410 415
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
420 425 430
Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys 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 Cys 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
<210> 101
<211> 227
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCL
<400> 101
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
115 120 125
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
130 135 140
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
145 150 155 160
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
165 170 175
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
180 185 190
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
195 200 205
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
210 215 220
Gly Glu Cys
225
<210> 102
<211> 697
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH1 "EE"- Fc PGLALA CEA VHCL
<400> 102
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Val Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr
515 520 525
Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
530 535 540
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
545 550 555 560
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr
565 570 575
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser
580 585 590
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
595 600 605
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
610 615 620
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
625 630 635 640
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
645 650 655
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
660 665 670
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
675 680 685
Thr Lys Ser Phe Asn Arg Gly Glu Cys
690 695
<210> 103
<211> 590
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH1- Fc 杵 CEA VH
<400> 103
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Ala Ala 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 Gly 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 Cys 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Val Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr
515 520 525
Thr Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
530 535 540
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
545 550 555 560
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr
565 570 575
Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
580 585 590
<210> 104
<211> 585
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH1- Fc 臼 CEA VL
<400> 104
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser
465 470 475 480
Ala Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly
485 490 495
Ile Asn Val Gly Ala Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly
500 505 510
Ser Pro Pro Gln Tyr Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln
515 520 525
Gln Gly Ser Gly Val Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser
530 535 540
Ala Asn Ala Gly Ile Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu
545 550 555 560
Ala Asp Tyr Tyr Cys Met Ile Trp His Ser Gly Ala Ser Ala Val Phe
565 570 575
Gly Gly Gly Thr Lys Leu Thr Val Leu
580 585
<210> 105
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VHCH1 "EE"- Fc 臼 PGLALA HYRF
<400> 105
Glu Val Lys Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ala Gly Gly Gly Thr Asn Tyr Asn Ser Ala Leu Met
50 55 60
Ser Arg Lys Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Asp Lys Gly Tyr Ser Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser 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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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
<210> 106
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> CD28(mAb 9.3) VLCL "RK"
<400> 106
Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Val Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Ser Leu Asn Ile His
65 70 75 80
Pro Val Asp Glu Asp Asp Val Ala Met Tyr Phe Cys Gln Gln Ser Arg
85 90 95
Lys Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 107
<211> 834
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1 "EE" Fc 臼 PGLALA FAP(4B9) VH - CEA(Medi-565)
VHCL
<400> 107
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
515 520 525
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
530 535 540
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
545 550 555 560
Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp
565 570 575
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
580 585 590
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
595 600 605
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu
610 615 620
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr Trp Met
625 630 635 640
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Phe
645 650 655
Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala Ser Val
660 665 670
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr
675 680 685
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
690 695 700
Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
705 710 715 720
Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe
725 730 735
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
740 745 750
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
755 760 765
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
770 775 780
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
785 790 795 800
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
805 810 815
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
820 825 830
Glu Cys
<210> 108
<211> 577
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1 "EE" Fc 杵 PGLALA FAP(4B9) VL
<400> 108
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
465 470 475 480
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
485 490 495
Ser Val Thr Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
500 505 510
Ala Pro Arg Leu Leu Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile
515 520 525
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
530 535 540
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
545 550 555 560
Gly Ile Met Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
565 570 575
Lys
<210> 109
<211> 221
<212> PRT
<213> 人工序列
<220>
<223> CEA VLCH1
<400> 109
Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala
1 5 10 15
Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly Ile Asn Val Gly Ala
20 25 30
Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr
35 40 45
Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val
50 55 60
Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile
65 70 75 80
Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Met Ile Trp His Ser Gly Ala Ser Ala Val Phe Gly Gly Gly Thr Lys
100 105 110
Leu Thr Val Leu 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
210 215 220
<210> 110
<211> 727
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1 Fc 臼 PGLALA FAP(4B9) VH - CEA VH
<400> 110
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly 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 Cys 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
465 470 475 480
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
485 490 495
Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
500 505 510
Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
515 520 525
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
530 535 540
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
545 550 555 560
Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp
565 570 575
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
580 585 590
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
595 600 605
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu
610 615 620
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr Trp Met
625 630 635 640
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Phe
645 650 655
Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala Ser Val
660 665 670
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr
675 680 685
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
690 695 700
Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
705 710 715 720
Thr Thr Val Thr Val Ser Ser
725
<210> 111
<211> 713
<212> PRT
<213> 人工序列
<220>
<223> CD28(SA) VHCH1 Fc 杵 PGLALA FAP(4B9) VL - CEA VL
<400> 111
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Ala Ala 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 Gly Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
465 470 475 480
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
485 490 495
Ser Val Thr Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
500 505 510
Ala Pro Arg Leu Leu Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile
515 520 525
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
530 535 540
Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
545 550 555 560
Gly Ile Met Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
565 570 575
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
580 585 590
Gly Gly Gly Gly Ser Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser
595 600 605
Ala Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly
610 615 620
Ile Asn Val Gly Ala Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly
625 630 635 640
Ser Pro Pro Gln Tyr Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln
645 650 655
Gln Gly Ser Gly Val Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser
660 665 670
Ala Asn Ala Gly Ile Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu
675 680 685
Ala Asp Tyr Tyr Cys Met Ile Trp His Ser Gly Ala Ser Ala Val Phe
690 695 700
Gly Gly Gly Thr Lys Leu Thr Val Leu
705 710
<210> 112
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 亲代)CH1- Fc 杵 PGLALA
<400> 112
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 113
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 g)CH1- Fc 杵 PGLALA
<400> 113
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Arg Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp His Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 114
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 f)CH1- Fc 杵 PGLALA
<400> 114
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Phe Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 115
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 j)CH1- Fc 杵 PGLALA
<400> 115
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 Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Tyr Pro Gly Asn Val Ala Thr Arg 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
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 116
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 e)CH1- Fc 杵 PGLALA
<400> 116
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 117
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 b)CH1- Fc 杵 PGLALA
<400> 117
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Gln Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp His Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 118
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 a)CH1- Fc 杵 PGLALA
<400> 118
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 Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 119
<211> 448
<212> PRT
<213> 人工序列
<220>
<223> VH(CD28 变体 i)CH1- Fc 杵 PGLALA
<400> 119
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 Thr Ser Tyr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Tyr Pro Gly Asn Val Asn Thr Arg 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
Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln
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 Glu 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 Glu Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 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 Gly 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 Cys 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
<210> 120
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> VL(CD28 变体 k)-CL
<400> 120
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 His Ala Ser Gln Asn Ile Tyr Val His
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Ala Gln Thr Tyr Pro Tyr
85 90 95
Thr 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 Arg Lys 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> 121
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> VL(CD28 变体 l)-CL
<400> 121
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 His Ala Ser Gln Asn Ile Tyr Val Phe
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr 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 Arg Lys 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> 122
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> VL(CD28 变体 m)-CL
<400> 122
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 His Ala Ser Gln Asn Ile Tyr Val Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr 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 Arg Lys 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> 123
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> VL(CD28 变体 r)-CL
<400> 123
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 His Ala Ser Gln Gly Ile Tyr Val Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr 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 Arg Lys 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> 124
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> VL(CD28 变体 s)-CL
<400> 124
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 His Ala Ser Gln Gly Ile Ser Val Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu His Thr 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr 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 Arg Lys 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> 125
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> VL(CD28 变体 t)-CL
<400> 125
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 Asn Ile Tyr Val Trp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Lys Ala Ser Asn Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg 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 Gly Gln Thr Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln 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 Arg Lys 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> 126
<211> 225
<212> PRT
<213> 人工序列
<220>
<223> Fc 臼 PGLALA、HYRF
<400> 126
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro
225
<210> 127
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> CEA CDR-H1
<400> 127
Ser Tyr Trp Met His
1 5
<210> 128
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> CEA CDR-H2
<400> 128
Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala Ser
1 5 10 15
Val Lys Gly
<210> 129
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> CEA CDR-H3
<400> 129
Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr
1 5 10
<210> 130
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> CEA CDR-L1
<400> 130
Thr Leu Arg Arg Gly Ile Asn Val Gly Ala Tyr Ser Ile Tyr
1 5 10
<210> 131
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> CEA CDR-L2
<400> 131
Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val
1 5 10
<210> 132
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> CEA CDR-L3
<400> 132
Met Ile Trp His Ser Gly Ala Ser Ala Val
1 5 10
<210> 133
<211> 121
<212> PRT
<213> 人工序列
<220>
<223> CEA VH
<400> 133
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 Val Ser Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Phe Ile Arg Asn Lys Ala Asn Gly Gly Thr Thr Glu Tyr Ala Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 134
<211> 116
<212> PRT
<213> 人工序列
<220>
<223> CEA VL
<400> 134
Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala
1 5 10 15
Ser Ala Ser Leu Thr Cys Thr Leu Arg Arg Gly Ile Asn Val Gly Ala
20 25 30
Tyr Ser Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr
35 40 45
Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val
50 55 60
Ser Ser Arg Phe Ser Ala Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile
65 70 75 80
Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Met Ile Trp His Ser Gly Ala Ser Ala Val Phe Gly Gly Gly Thr Lys
100 105 110
Leu Thr Val Leu
115
<210> 135
<211> 748
<212> PRT
<213> 人工序列
<220>
<223> His-tagged human FAP ECD
<400> 135
Arg Pro Ser Arg Val His Asn Ser Glu Glu Asn Thr Met Arg Ala Leu
1 5 10 15
Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe
20 25 30
Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala Asp Asn
35 40 45
Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu
50 55 60
Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser
65 70 75 80
Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp
85 90 95
Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly
100 105 110
Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys
115 120 125
Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile
130 135 140
Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn
145 150 155 160
Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu
165 170 175
Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly
180 185 190
Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val Ile
195 200 205
Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile
210 215 220
Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val Arg Ile Phe Ile
225 230 235 240
Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro Val
245 250 255
Pro Ala Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp
260 265 270
Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn
275 280 285
Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp
290 295 300
Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp
305 310 315 320
Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile
325 330 335
Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His
340 345 350
Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys
355 360 365
Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr
370 375 380
Ser Ser Asn Glu Phe Glu Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg
385 390 395 400
Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys His
405 410 415
Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr
420 425 430
Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser
435 440 445
Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu
450 455 460
Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu
465 470 475 480
Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met
485 490 495
Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile
500 505 510
Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala
515 520 525
Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala
530 535 540
Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr
545 550 555 560
Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr
565 570 575
Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile
580 585 590
Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu
595 600 605
Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val
610 615 620
Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly
625 630 635 640
Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val
645 650 655
Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His
660 665 670
Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala
675 680 685
Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser
690 695 700
Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr Thr
705 710 715 720
His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp Gly Lys
725 730 735
Lys Lys Lys Lys Lys Gly His His His His His His
740 745
<210> 136
<211> 761
<212> PRT
<213> 鼠
<400> 136
Met Lys Thr Trp Leu Lys Thr Val Phe Gly Val Thr Thr Leu Ala Ala
1 5 10 15
Leu Ala Leu Val Val Ile Cys Ile Val Leu Arg Pro Ser Arg Val Tyr
20 25 30
Lys Pro Glu Gly Asn Thr Lys Arg Ala Leu Thr Leu Lys Asp Ile Leu
35 40 45
Asn Gly Thr Phe Ser Tyr Lys Thr Tyr Phe Pro Asn Trp Ile Ser Glu
50 55 60
Gln Glu Tyr Leu His Gln Ser Glu Asp Asp Asn Ile Val Phe Tyr Asn
65 70 75 80
Ile Glu Thr Arg Glu Ser Tyr Ile Ile Leu Ser Asn Ser Thr Met Lys
85 90 95
Ser Val Asn Ala Thr Asp Tyr Gly Leu Ser Pro Asp Arg Gln Phe Val
100 105 110
Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala
115 120 125
Thr Tyr Tyr Ile Tyr Asp Leu Gln Asn Gly Glu Phe Val Arg Gly Tyr
130 135 140
Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser
145 150 155 160
Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro
165 170 175
Gly Asp Pro Pro Phe Gln Ile Thr Tyr Thr Gly Arg Glu Asn Arg Ile
180 185 190
Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu Ala Thr
195 200 205
Lys Tyr Ala Leu Trp Trp Ser Pro Asp Gly Lys Phe Leu Ala Tyr Val
210 215 220
Glu Phe Asn Asp Ser Asp Ile Pro Ile Ile Ala Tyr Ser Tyr Tyr Gly
225 230 235 240
Asp Gly Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly
245 250 255
Ala Lys Asn Pro Val Val Arg Val Phe Ile Val Asp Thr Thr Tyr Pro
260 265 270
His His Val Gly Pro Met Glu Val Pro Val Pro Glu Met Ile Ala Ser
275 280 285
Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Ser Ser Glu Arg Val
290 295 300
Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu Ser Ile
305 310 315 320
Cys Asp Phe Arg Glu Asp Trp His Ala Trp Glu Cys Pro Lys Asn Gln
325 330 335
Glu His Val Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val
340 345 350
Ser Thr Pro Ala Phe Ser Gln Asp Ala Thr Ser Tyr Tyr Lys Ile Phe
355 360 365
Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val
370 375 380
Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Tyr Ile
385 390 395 400
Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu
405 410 415
Gly Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Asn Ser
420 425 430
Pro Pro Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg Cys
435 440 445
Gln Tyr Tyr Thr Ala Ser Phe Ser Tyr Lys Ala Lys Tyr Tyr Ala Leu
450 455 460
Val Cys Tyr Gly Pro Gly Leu Pro Ile Ser Thr Leu His Asp Gly Arg
465 470 475 480
Thr Asp Gln Glu Ile Gln Val Leu Glu Glu Asn Lys Glu Leu Glu Asn
485 490 495
Ser Leu Arg Asn Ile Gln Leu Pro Lys Val Glu Ile Lys Lys Leu Lys
500 505 510
Asp Gly Gly Leu Thr Phe Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe
515 520 525
Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro
530 535 540
Cys Ser Gln Ser Val Lys Ser Val Phe Ala Val Asn Trp Ile Thr Tyr
545 550 555 560
Leu Ala Ser Lys Glu Gly Ile Val Ile Ala Leu Val Asp Gly Arg Gly
565 570 575
Thr Ala Phe Gln Gly Asp Lys Phe Leu His Ala Val Tyr Arg Lys Leu
580 585 590
Gly Val Tyr Glu Val Glu Asp Gln Leu Thr Ala Val Arg Lys Phe Ile
595 600 605
Glu Met Gly Phe Ile Asp Glu Glu Arg Ile Ala Ile Trp Gly Trp Ser
610 615 620
Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu Ala Ser Gly Thr Gly Leu
625 630 635 640
Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr
645 650 655
Ala Ser Ile Tyr Ser Glu Arg Phe Met Gly Leu Pro Thr Lys Asp Asp
660 665 670
Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala Glu Tyr
675 680 685
Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp Asp Asn
690 695 700
Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val Asn Ala
705 710 715 720
Gln Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His Gly Ile
725 730 735
Ser Ser Gly Arg Ser Gln Asn His Leu Tyr Thr His Met Thr His Phe
740 745 750
Leu Lys Gln Cys Phe Ser Leu Ser Asp
755 760
<210> 137
<211> 749
<212> PRT
<213> 人工序列
<220>
<223> 鼠 FAP 胞外域 + 聚-lys-标签 + his6-标签
<400> 137
Arg Pro Ser Arg Val Tyr Lys Pro Glu Gly Asn Thr Lys Arg Ala Leu
1 5 10 15
Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Tyr Phe
20 25 30
Pro Asn Trp Ile Ser Glu Gln Glu Tyr Leu His Gln Ser Glu Asp Asp
35 40 45
Asn Ile Val Phe Tyr Asn Ile Glu Thr Arg Glu Ser Tyr Ile Ile Leu
50 55 60
Ser Asn Ser Thr Met Lys Ser Val Asn Ala Thr Asp Tyr Gly Leu Ser
65 70 75 80
Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp
85 90 95
Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Gln Asn Gly
100 105 110
Glu Phe Val Arg Gly Tyr Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys
115 120 125
Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile
130 135 140
Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Tyr Thr
145 150 155 160
Gly Arg Glu Asn Arg Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu
165 170 175
Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asp Gly
180 185 190
Lys Phe Leu Ala Tyr Val Glu Phe Asn Asp Ser Asp Ile Pro Ile Ile
195 200 205
Ala Tyr Ser Tyr Tyr Gly Asp Gly Gln Tyr Pro Arg Thr Ile Asn Ile
210 215 220
Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val Arg Val Phe Ile
225 230 235 240
Val Asp Thr Thr Tyr Pro His His Val Gly Pro Met Glu Val Pro Val
245 250 255
Pro Glu Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp
260 265 270
Val Ser Ser Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn
275 280 285
Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp His Ala Trp
290 295 300
Glu Cys Pro Lys Asn Gln Glu His Val Glu Glu Ser Arg Thr Gly Trp
305 310 315 320
Ala Gly Gly Phe Phe Val Ser Thr Pro Ala Phe Ser Gln Asp Ala Thr
325 330 335
Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His
340 345 350
Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys
355 360 365
Trp Glu Ala Ile Tyr Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr
370 375 380
Ser Ser Asn Glu Phe Glu Gly Tyr Pro Gly Arg Arg Asn Ile Tyr Arg
385 390 395 400
Ile Ser Ile Gly Asn Ser Pro Pro Ser Lys Lys Cys Val Thr Cys His
405 410 415
Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Tyr Lys
420 425 430
Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Leu Pro Ile Ser
435 440 445
Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Gln Val Leu Glu Glu
450 455 460
Asn Lys Glu Leu Glu Asn Ser Leu Arg Asn Ile Gln Leu Pro Lys Val
465 470 475 480
Glu Ile Lys Lys Leu Lys Asp Gly Gly Leu Thr Phe Trp Tyr Lys Met
485 490 495
Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile
500 505 510
Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Lys Ser Val Phe Ala
515 520 525
Val Asn Trp Ile Thr Tyr Leu Ala Ser Lys Glu Gly Ile Val Ile Ala
530 535 540
Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Phe Leu His
545 550 555 560
Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Leu Thr
565 570 575
Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Glu Arg Ile
580 585 590
Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu
595 600 605
Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val
610 615 620
Ser Ser Trp Glu Tyr Tyr Ala Ser Ile Tyr Ser Glu Arg Phe Met Gly
625 630 635 640
Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val
645 650 655
Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His
660 665 670
Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala
675 680 685
Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser
690 695 700
Asp Gln Asn His Gly Ile Leu Ser Gly Arg Ser Gln Asn His Leu Tyr
705 710 715 720
Thr His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp Gly
725 730 735
Lys Lys Lys Lys Lys Lys Gly His His His His His His
740 745
<210> 138
<211> 748
<212> PRT
<213> 人工序列
<220>
<223> 食蟹猴 FAP 胞外域 + 聚-lys-标签 + his6-标签
<400> 138
Arg Pro Pro Arg Val His Asn Ser Glu Glu Asn Thr Met Arg Ala Leu
1 5 10 15
Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe
20 25 30
Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala Asp Asn
35 40 45
Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu
50 55 60
Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser
65 70 75 80
Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp
85 90 95
Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly
100 105 110
Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys
115 120 125
Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile
130 135 140
Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn
145 150 155 160
Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu
165 170 175
Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly
180 185 190
Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val Ile
195 200 205
Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile
210 215 220
Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Phe Val Arg Ile Phe Ile
225 230 235 240
Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro Val
245 250 255
Pro Ala Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp
260 265 270
Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn
275 280 285
Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp
290 295 300
Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp
305 310 315 320
Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile
325 330 335
Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His
340 345 350
Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys
355 360 365
Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr
370 375 380
Ser Ser Asn Glu Phe Glu Asp Tyr Pro Gly Arg Arg Asn Ile Tyr Arg
385 390 395 400
Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys His
405 410 415
Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr
420 425 430
Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser
435 440 445
Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu
450 455 460
Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu
465 470 475 480
Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met
485 490 495
Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile
500 505 510
Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala
515 520 525
Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala
530 535 540
Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr
545 550 555 560
Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr
565 570 575
Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile
580 585 590
Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu
595 600 605
Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val
610 615 620
Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly
625 630 635 640
Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val
645 650 655
Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His
660 665 670
Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala
675 680 685
Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser
690 695 700
Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr Thr
705 710 715 720
His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp Gly Lys
725 730 735
Lys Lys Lys Lys Lys Gly His His His His His His
740 745
<210> 139
<211> 257
<212> PRT
<213> 智人
<400> 139
Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val
1 5 10 15
Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu
20 25 30
Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly
35 40 45
Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala
50 55 60
Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr
65 70 75 80
Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys
85 90 95
Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn
100 105 110
Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg
115 120 125
Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu
130 135 140
Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp
145 150 155 160
Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln
165 170 175
Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile
180 185 190
Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg
195 200 205
Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu
210 215 220
Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala
225 230 235 240
Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu
245 250 255
Ser
<210> 140
<211> 255
<212> PRT
<213> 小家鼠
<400> 140
Met Ala His Leu Met Thr Val Gln Leu Leu Leu Leu Val Met Trp Met
1 5 10 15
Ala Glu Cys Ala Gln Ser Arg Ala Thr Arg Ala Arg Thr Glu Leu Leu
20 25 30
Asn Val Cys Met Asp Ala Lys His His Lys Glu Lys Pro Gly Pro Glu
35 40 45
Asp Asn Leu His Asp Gln Cys Ser Pro Trp Lys Thr Asn Ser Cys Cys
50 55 60
Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Ile Ser Tyr Leu Tyr
65 70 75 80
Arg Phe Asn Trp Asn His Cys Gly Thr Met Thr Ser Glu Cys Lys Arg
85 90 95
His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn Leu Gly
100 105 110
Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg Ile Leu
115 120 125
Asp Val Pro Leu Cys Lys Glu Asp Cys Gln Gln Trp Trp Glu Asp Cys
130 135 140
Gln Ser Ser Phe Thr Cys Lys Ser Asn Trp His Lys Gly Trp Asn Trp
145 150 155 160
Ser Ser Gly His Asn Glu Cys Pro Val Gly Ala Ser Cys His Pro Phe
165 170 175
Thr Phe Tyr Phe Pro Thr Ser Ala Ala Leu Cys Glu Glu Ile Trp Ser
180 185 190
His Ser Tyr Lys Leu Ser Asn Tyr Ser Arg Gly Ser Gly Arg Cys Ile
195 200 205
Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu Val Ala
210 215 220
Arg Phe Tyr Ala Glu Ala Met Ser Gly Ala Gly Phe His Gly Thr Trp
225 230 235 240
Pro Leu Leu Cys Ser Leu Ser Leu Val Leu Leu Trp Val Ile Ser
245 250 255
<210> 141
<211> 257
<212> PRT
<213> 食蟹猴
<400> 141
Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val
1 5 10 15
Ala Val Val Gly Glu Ala Gln Thr Arg Thr Ala Arg Ala Arg Thr Glu
20 25 30
Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly
35 40 45
Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Lys Lys Asn Ala
50 55 60
Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr
65 70 75 80
Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys
85 90 95
Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn
100 105 110
Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg
115 120 125
Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Arg Trp Trp Glu
130 135 140
Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp
145 150 155 160
Asn Trp Thr Ser Gly Phe Asn Lys Cys Pro Val Gly Ala Ala Cys Gln
165 170 175
Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile
180 185 190
Trp Thr Tyr Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg
195 200 205
Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu
210 215 220
Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala
225 230 235 240
Ala Trp Pro Leu Leu Leu Ser Leu Ala Leu Thr Leu Leu Trp Leu Leu
245 250 255
Ser
<210> 142
<211> 2322
<212> PRT
<213> 智人
<400> 142
Met Gln Ser Gly Pro Arg Pro Pro Leu Pro Ala Pro Gly Leu Ala Leu
1 5 10 15
Ala Leu Thr Leu Thr Met Leu Ala Arg Leu Ala Ser Ala Ala Ser Phe
20 25 30
Phe Gly Glu Asn His Leu Glu Val Pro Val Ala Thr Ala Leu Thr Asp
35 40 45
Ile Asp Leu Gln Leu Gln Phe Ser Thr Ser Gln Pro Glu Ala Leu Leu
50 55 60
Leu Leu Ala Ala Gly Pro Ala Asp His Leu Leu Leu Gln Leu Tyr Ser
65 70 75 80
Gly Arg Leu Gln Val Arg Leu Val Leu Gly Gln Glu Glu Leu Arg Leu
85 90 95
Gln Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser Ile Pro His Thr Val
100 105 110
Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe
115 120 125
Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro Tyr
130 135 140
Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro Tyr Leu Arg
145 150 155 160
Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu His Ala Ala Thr Leu Asn
165 170 175
Gly Arg Ser Leu Leu Arg Pro Leu Thr Pro Asp Val His Glu Gly Cys
180 185 190
Ala Glu Glu Phe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly
195 200 205
Pro His Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gln Asp Glu Gly
210 215 220
Thr Leu Glu Phe Thr Leu Thr Thr Gln Ser Arg Gln Ala Pro Leu Ala
225 230 235 240
Phe Gln Ala Gly Gly Arg Arg Gly Asp Phe Ile Tyr Val Asp Ile Phe
245 250 255
Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gln Gly Thr Val Leu
260 265 270
Leu His Asn Ser Val Pro Val Ala Asp Gly Gln Pro His Glu Val Ser
275 280 285
Val His Ile Asn Ala His Arg Leu Glu Ile Ser Val Asp Gln Tyr Pro
290 295 300
Thr His Thr Ser Asn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg Gly
305 310 315 320
Ser Leu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gln
325 330 335
Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu
340 345 350
Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gln Arg Arg Gly Leu Arg
355 360 365
Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg Leu Glu Glu
370 375 380
Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr Glu Ala Phe Ser Thr
385 390 395 400
Leu Ala Pro Glu Ala Trp Pro Ala Met Glu Leu Pro Glu Pro Cys Val
405 410 415
Pro Glu Pro Gly Leu Pro Pro Val Phe Ala Asn Phe Thr Gln Leu Leu
420 425 430
Thr Ile Ser Pro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu
435 440 445
Trp Arg His Val Gln Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg
450 455 460
Lys Ser Gln Val Leu Phe Ser Val Thr Arg Gly Ala Arg His Gly Glu
465 470 475 480
Leu Glu Leu Asp Ile Pro Gly Ala Gln Ala Arg Lys Met Phe Thr Leu
485 490 495
Leu Asp Val Val Asn Arg Lys Ala Arg Phe Ile His Asp Gly Ser Glu
500 505 510
Asp Thr Ser Asp Gln Leu Val Leu Glu Val Ser Val Thr Ala Arg Val
515 520 525
Pro Met Pro Ser Cys Leu Arg Arg Gly Gln Thr Tyr Leu Leu Pro Ile
530 535 540
Gln Val Asn Pro Val Asn Asp Pro Pro His Ile Ile Phe Pro His Gly
545 550 555 560
Ser Leu Met Val Ile Leu Glu His Thr Gln Lys Pro Leu Gly Pro Glu
565 570 575
Val Phe Gln Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe
580 585 590
Gln Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg Arg Asp Gln
595 600 605
Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg Glu Leu Glu Ala Gly
610 615 620
Ser Leu Val Tyr Val His Arg Gly Gly Pro Ala Gln Asp Leu Thr Phe
625 630 635 640
Arg Val Ser Asp Gly Leu Gln Ala Ser Pro Pro Ala Thr Leu Lys Val
645 650 655
Val Ala Ile Arg Pro Ala Ile Gln Ile His Arg Ser Thr Gly Leu Arg
660 665 670
Leu Ala Gln Gly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val
675 680 685
Glu Thr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr
690 695 700
Gly Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala Gly Gly Val
705 710 715 720
Glu Gly Ala Glu Trp Trp Ala Thr Gln Ala Phe His Gln Arg Asp Val
725 730 735
Glu Gln Gly Arg Val Arg Tyr Leu Ser Thr Asp Pro Gln His His Ala
740 745 750
Tyr Asp Thr Val Glu Asn Leu Ala Leu Glu Val Gln Val Gly Gln Glu
755 760 765
Ile Leu Ser Asn Leu Ser Phe Pro Val Thr Ile Gln Arg Ala Thr Val
770 775 780
Trp Met Leu Arg Leu Glu Pro Leu His Thr Gln Asn Thr Gln Gln Glu
785 790 795 800
Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro
805 810 815
Ser Pro Pro Thr Phe His Tyr Glu Val Val Gln Ala Pro Arg Lys Gly
820 825 830
Asn Leu Gln Leu Gln Gly Thr Arg Leu Ser Asp Gly Gln Gly Phe Thr
835 840 845
Gln Asp Asp Ile Gln Ala Gly Arg Val Thr Tyr Gly Ala Thr Ala Arg
850 855 860
Ala Ser Glu Ala Val Glu Asp Thr Phe Arg Phe Arg Val Thr Ala Pro
865 870 875 880
Pro Tyr Phe Ser Pro Leu Tyr Thr Phe Pro Ile His Ile Gly Gly Asp
885 890 895
Pro Asp Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Gly
900 905 910
Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn
915 920 925
Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg His Gly Arg
930 935 940
Leu Ala Trp Arg Gly Thr Gln Asp Lys Thr Thr Met Val Thr Ser Phe
945 950 955 960
Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val Tyr Gln His Asp Asp
965 970 975
Ser Glu Thr Thr Glu Asp Asp Ile Pro Phe Val Ala Thr Arg Gln Gly
980 985 990
Glu Ser Ser Gly Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg
995 1000 1005
Val Ala Ile Gln Pro Val Asn Asp His Ala Pro Val Gln Thr Ile
1010 1015 1020
Ser Arg Ile Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr
1025 1030 1035
Thr Asp Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp
1040 1045 1050
Ala Gln Leu Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser Ile
1055 1060 1065
Val Ala Val Asp Glu Pro Thr Arg Pro Ile Tyr Arg Phe Thr Gln
1070 1075 1080
Glu Asp Leu Arg Lys Arg Arg Val Leu Phe Val His Ser Gly Ala
1085 1090 1095
Asp Arg Gly Trp Ile Gln Leu Gln Val Ser Asp Gly Gln His Gln
1100 1105 1110
Ala Thr Ala Leu Leu Glu Val Gln Ala Ser Glu Pro Tyr Leu Arg
1115 1120 1125
Val Ala Asn Gly Ser Ser Leu Val Val Pro Gln Gly Gly Gln Gly
1130 1135 1140
Thr Ile Asp Thr Ala Val Leu His Leu Asp Thr Asn Leu Asp Ile
1145 1150 1155
Arg Ser Gly Asp Glu Val His Tyr His Val Thr Ala Gly Pro Arg
1160 1165 1170
Trp Gly Gln Leu Val Arg Ala Gly Gln Pro Ala Thr Ala Phe Ser
1175 1180 1185
Gln Gln Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser His Asn Gly
1190 1195 1200
Ser Leu Ser Pro Arg Asp Thr Met Ala Phe Ser Val Glu Ala Gly
1205 1210 1215
Pro Val His Thr Asp Ala Thr Leu Gln Val Thr Ile Ala Leu Glu
1220 1225 1230
Gly Pro Leu Ala Pro Leu Lys Leu Val Arg His Lys Lys Ile Tyr
1235 1240 1245
Val Phe Gln Gly Glu Ala Ala Glu Ile Arg Arg Asp Gln Leu Glu
1250 1255 1260
Ala Ala Gln Glu Ala Val Pro Pro Ala Asp Ile Val Phe Ser Val
1265 1270 1275
Lys Ser Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly
1280 1285 1290
Ala Leu Ala Asp Glu Pro Pro Ser Leu Asp Pro Val Gln Ser Phe
1295 1300 1305
Ser Gln Glu Ala Val Asp Thr Gly Arg Val Leu Tyr Leu His Ser
1310 1315 1320
Arg Pro Glu Ala Trp Ser Asp Ala Phe Ser Leu Asp Val Ala Ser
1325 1330 1335
Gly Leu Gly Ala Pro Leu Glu Gly Val Leu Val Glu Leu Glu Val
1340 1345 1350
Leu Pro Ala Ala Ile Pro Leu Glu Ala Gln Asn Phe Ser Val Pro
1355 1360 1365
Glu Gly Gly Ser Leu Thr Leu Ala Pro Pro Leu Leu Arg Val Ser
1370 1375 1380
Gly Pro Tyr Phe Pro Thr Leu Leu Gly Leu Ser Leu Gln Val Leu
1385 1390 1395
Glu Pro Pro Gln His Gly Ala Leu Gln Lys Glu Asp Gly Pro Gln
1400 1405 1410
Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met Val Glu Glu Gln
1415 1420 1425
Leu Ile Arg Tyr Val His Asp Gly Ser Glu Thr Leu Thr Asp Ser
1430 1435 1440
Phe Val Leu Met Ala Asn Ala Ser Glu Met Asp Arg Gln Ser His
1445 1450 1455
Pro Val Ala Phe Thr Val Thr Val Leu Pro Val Asn Asp Gln Pro
1460 1465 1470
Pro Ile Leu Thr Thr Asn Thr Gly Leu Gln Met Trp Glu Gly Ala
1475 1480 1485
Thr Ala Pro Ile Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp
1490 1495 1500
Ser Gly Ser Glu Asp Leu Val Tyr Thr Ile Glu Gln Pro Ser Asn
1505 1510 1515
Gly Arg Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser
1520 1525 1530
Phe Thr Gln Ala Gln Leu Asp Gly Gly Leu Val Leu Phe Ser His
1535 1540 1545
Arg Gly Thr Leu Asp Gly Gly Phe Arg Phe Arg Leu Ser Asp Gly
1550 1555 1560
Glu His Thr Ser Pro Gly His Phe Phe Arg Val Thr Ala Gln Lys
1565 1570 1575
Gln Val Leu Leu Ser Leu Lys Gly Ser Gln Thr Leu Thr Val Cys
1580 1585 1590
Pro Gly Ser Val Gln Pro Leu Ser Ser Gln Thr Leu Arg Ala Ser
1595 1600 1605
Ser Ser Ala Gly Thr Asp Pro Gln Leu Leu Leu Tyr Arg Val Val
1610 1615 1620
Arg Gly Pro Gln Leu Gly Arg Leu Phe His Ala Gln Gln Asp Ser
1625 1630 1635
Thr Gly Glu Ala Leu Val Asn Phe Thr Gln Ala Glu Val Tyr Ala
1640 1645 1650
Gly Asn Ile Leu Tyr Glu His Glu Met Pro Pro Glu Pro Phe Trp
1655 1660 1665
Glu Ala His Asp Thr Leu Glu Leu Gln Leu Ser Ser Pro Pro Ala
1670 1675 1680
Arg Asp Val Ala Ala Thr Leu Ala Val Ala Val Ser Phe Glu Ala
1685 1690 1695
Ala Cys Pro Gln Arg Pro Ser His Leu Trp Lys Asn Lys Gly Leu
1700 1705 1710
Trp Val Pro Glu Gly Gln Arg Ala Arg Ile Thr Val Ala Ala Leu
1715 1720 1725
Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gln Arg Ser
1730 1735 1740
Glu His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Ser Arg Gly
1745 1750 1755
Gln Leu Leu Val Ser Glu Glu Pro Leu His Ala Gly Gln Pro His
1760 1765 1770
Phe Leu Gln Ser Gln Leu Ala Ala Gly Gln Leu Val Tyr Ala His
1775 1780 1785
Gly Gly Gly Gly Thr Gln Gln Asp Gly Phe His Phe Arg Ala His
1790 1795 1800
Leu Gln Gly Pro Ala Gly Ala Ser Val Ala Gly Pro Gln Thr Ser
1805 1810 1815
Glu Ala Phe Ala Ile Thr Val Arg Asp Val Asn Glu Arg Pro Pro
1820 1825 1830
Gln Pro Gln Ala Ser Val Pro Leu Arg Leu Thr Arg Gly Ser Arg
1835 1840 1845
Ala Pro Ile Ser Arg Ala Gln Leu Ser Val Val Asp Pro Asp Ser
1850 1855 1860
Ala Pro Gly Glu Ile Glu Tyr Glu Val Gln Arg Ala Pro His Asn
1865 1870 1875
Gly Phe Leu Ser Leu Val Gly Gly Gly Leu Gly Pro Val Thr Arg
1880 1885 1890
Phe Thr Gln Ala Asp Val Asp Ser Gly Arg Leu Ala Phe Val Ala
1895 1900 1905
Asn Gly Ser Ser Val Ala Gly Ile Phe Gln Leu Ser Met Ser Asp
1910 1915 1920
Gly Ala Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp Ile Leu
1925 1930 1935
Pro Ser Ala Ile Glu Val Gln Leu Arg Ala Pro Leu Glu Val Pro
1940 1945 1950
Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln Gln Gln Leu Arg Val
1955 1960 1965
Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu Ile Gln
1970 1975 1980
Gly Pro Gln Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser
1985 1990 1995
Ala Phe Ser Gln Phe Gln Ile Asp Gln Gly Glu Val Val Phe Ala
2000 2005 2010
Phe Thr Asn Phe Ser Ser Ser His Asp His Phe Arg Val Leu Ala
2015 2020 2025
Leu Ala Arg Gly Val Asn Ala Ser Ala Val Val Asn Val Thr Val
2030 2035 2040
Arg Ala Leu Leu His Val Trp Ala Gly Gly Pro Trp Pro Gln Gly
2045 2050 2055
Ala Thr Leu Arg Leu Asp Pro Thr Val Leu Asp Ala Gly Glu Leu
2060 2065 2070
Ala Asn Arg Thr Gly Ser Val Pro Arg Phe Arg Leu Leu Glu Gly
2075 2080 2085
Pro Arg His Gly Arg Val Val Arg Val Pro Arg Ala Arg Thr Glu
2090 2095 2100
Pro Gly Gly Ser Gln Leu Val Glu Gln Phe Thr Gln Gln Asp Leu
2105 2110 2115
Glu Asp Gly Arg Leu Gly Leu Glu Val Gly Arg Pro Glu Gly Arg
2120 2125 2130
Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu Glu Leu Trp Ala
2135 2140 2145
Gln Gly Val Pro Pro Ala Val Ala Ser Leu Asp Phe Ala Thr Glu
2150 2155 2160
Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu Ser Val
2165 2170 2175
Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser Thr
2180 2185 2190
Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala
2195 2200 2205
Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe
2210 2215 2220
Ser Val Ile Ile Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu
2225 2230 2235
Ile Leu Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys Thr Gly
2240 2245 2250
Lys His Asp Val Gln Val Leu Thr Ala Lys Pro Arg Asn Gly Leu
2255 2260 2265
Ala Gly Asp Thr Glu Thr Phe Arg Lys Val Glu Pro Gly Gln Ala
2270 2275 2280
Ile Pro Leu Thr Ala Val Pro Gly Gln Gly Pro Pro Pro Gly Gly
2285 2290 2295
Gln Pro Asp Pro Glu Leu Leu Gln Phe Cys Arg Thr Pro Asn Pro
2300 2305 2310
Ala Leu Lys Asn Gly Gln Tyr Trp Val
2315 2320
<210> 143
<211> 1210
<212> PRT
<213> 智人
<400> 143
Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala
1 5 10 15
Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn
50 55 60
Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys
65 70 75 80
Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val
85 90 95
Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr
100 105 110
Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn
115 120 125
Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu
130 135 140
His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu
145 150 155 160
Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met
165 170 175
Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro
180 185 190
Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln
195 200 205
Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg
210 215 220
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys
225 230 235 240
Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp
245 250 255
Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro
260 265 270
Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly
275 280 285
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
290 295 300
Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu
305 310 315 320
Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val
325 330 335
Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp
355 360 365
Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr
370 375 380
Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu
385 390 395 400
Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp
405 410 415
Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln
420 425 430
His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu
435 440 445
Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser
450 455 460
Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu
465 470 475 480
Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu
485 490 495
Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
500 505 510
Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn
515 520 525
Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
545 550 555 560
Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro
565 570 575
Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val
580 585 590
Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
595 600 605
Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys
610 615 620
Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly
625 630 635 640
Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu
645 650 655
Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His
660 665 670
Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu
675 680 685
Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu
690 695 700
Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser
705 710 715 720
Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu
725 730 735
Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser
740 745 750
Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser
755 760 765
Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser
770 775 780
Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp
785 790 795 800
Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
805 810 815
Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg
820 825 830
Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro
835 840 845
Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala
850 855 860
Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp
865 870 875 880
Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp
885 890 895
Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser
900 905 910
Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu
915 920 925
Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr
930 935 940
Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys
945 950 955 960
Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln
965 970 975
Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro
980 985 990
Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp
995 1000 1005
Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe
1010 1015 1020
Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu
1025 1030 1035
Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn
1040 1045 1050
Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg
1055 1060 1065
Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp
1070 1075 1080
Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro
1085 1090 1095
Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln
1100 1105 1110
Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro
1115 1120 1125
His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln
1130 1135 1140
Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala
1145 1150 1155
Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln
1160 1165 1170
Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys
1175 1180 1185
Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln
1190 1195 1200
Ser Ser Glu Phe Ile Gly Ala
1205 1210
<210> 144
<211> 1255
<212> PRT
<213> 智人 (Homo sapiens)
<400> 144
Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu
1 5 10 15
Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys
20 25 30
Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His
35 40 45
Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr
50 55 60
Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val
65 70 75 80
Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu
85 90 95
Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr
100 105 110
Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro
115 120 125
Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser
130 135 140
Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln
145 150 155 160
Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn
165 170 175
Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys
180 185 190
His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser
195 200 205
Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys
210 215 220
Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys
225 230 235 240
Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu
245 250 255
His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val
260 265 270
Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
275 280 285
Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu
290 295 300
Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln
305 310 315 320
Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys
325 330 335
Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu
340 345 350
Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys
355 360 365
Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp
370 375 380
Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe
385 390 395 400
Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro
405 410 415
Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
420 425 430
Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu
435 440 445
Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly
450 455 460
Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val
465 470 475 480
Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr
485 490 495
Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His
500 505 510
Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys
515 520 525
Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys
530 535 540
Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys
545 550 555 560
Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys
565 570 575
Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp
580 585 590
Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu
595 600 605
Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln
610 615 620
Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys
625 630 635 640
Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser
645 650 655
Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly
660 665 670
Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg
675 680 685
Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly
690 695 700
Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu
705 710 715 720
Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
725 730 735
Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile
740 745 750
Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu
755 760 765
Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg
770 775 780
Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu
785 790 795 800
Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg
805 810 815
Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly
820 825 830
Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala
835 840 845
Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe
850 855 860
Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp
865 870 875 880
Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg
885 890 895
Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val
900 905 910
Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala
915 920 925
Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro
930 935 940
Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met
945 950 955 960
Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe
965 970 975
Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu
980 985 990
Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu
995 1000 1005
Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr
1010 1015 1020
Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly
1025 1030 1035
Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg
1040 1045 1050
Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu
1055 1060 1065
Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser
1070 1075 1080
Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu
1085 1090 1095
Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser
1100 1105 1110
Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val
1115 1120 1125
Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro
1130 1135 1140
Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro
1145 1150 1155
Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu
1160 1165 1170
Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly
1175 1180 1185
Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala
1190 1195 1200
Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp
1205 1210 1215
Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro
1220 1225 1230
Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr
1235 1240 1245
Leu Gly Leu Asp Val Pro Val
1250 1255
<210> 145
<211> 645
<212> PRT
<213> 智人
<400> 145
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys
1 5 10 15
Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys
20 25 30
Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala Val
35 40 45
Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly Ile Leu
50 55 60
Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg Arg Leu
65 70 75 80
Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Ala Met
85 90 95
Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg Lys
100 105 110
Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile
115 120 125
Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Val
130 135 140
Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu
145 150 155 160
Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu Leu
165 170 175
Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu Met Pro
180 185 190
Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg Leu Gly
195 200 205
Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met Ser
210 215 220
Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn
225 230 235 240
Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu
245 250 255
Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly Gly
260 265 270
Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg
275 280 285
Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu
290 295 300
Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg Glu
305 310 315 320
Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile
325 330 335
Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp
340 345 350
Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg
355 360 365
Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp Leu
370 375 380
Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu Leu Glu
385 390 395 400
Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu Val Pro
405 410 415
Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly Gly Met
420 425 430
Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp
435 440 445
Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser Pro
450 455 460
Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu
465 470 475 480
Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His Asp Pro
485 490 495
Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu Pro Ser
500 505 510
Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu
515 520 525
Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu
530 535 540
Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro
545 550 555 560
Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala
565 570 575
Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly
580 585 590
Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp
595 600 605
Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro
610 615 620
Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu Gly
625 630 635 640
Leu Asp Val Pro Val
645
<210> 146
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基 G4S
<400> 146
Gly Gly Gly Gly Ser
1 5
<210> 147
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基 (G4S)2
<400> 147
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 148
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基 (SG4)2
<400> 148
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10
<210> 149
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基 G4(SG4)2
<400> 149
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10
<210> 150
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 150
Gly Ser Pro Gly Ser Ser Ser Ser Gly Ser
1 5 10
<210> 151
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基 (G4S)3
<400> 151
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 152
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基 (G4S)4
<400> 152
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser
20
<210> 153
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 153
Gly Ser Gly Ser Gly Ser Gly Ser
1 5
<210> 154
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 154
Gly Ser Gly Ser Gly Asn Gly Ser
1 5
<210> 155
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 155
Gly Gly Ser Gly Ser Gly Ser Gly
1 5
<210> 156
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 156
Gly Gly Ser Gly Ser Gly
1 5
<210> 157
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 157
Gly Gly Ser Gly
1
<210> 158
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 158
Gly Gly Ser Gly Asn Gly Ser Gly
1 5
<210> 159
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 159
Gly Gly Asn Gly Ser Gly Ser Gly
1 5
<210> 160
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 肽连接基
<400> 160
Gly Gly Asn Gly Ser Gly
1 5
<210> 161
<211> 420
<212> PRT
<213> 人工序列
<220>
<223> 基于 CEACAM5 的抗原 Hu N(A2-B2)A-avi-His
<400> 161
Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly Lys Glu
1 5 10 15
Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly Tyr Ser
20 25 30
Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val Gly Tyr
35 40 45
Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser Gly Arg
50 55 60
Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln
65 70 75 80
Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val
85 90 95
Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys
100 105 110
Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Glu Asp Ala
115 120 125
Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr Tyr Leu Trp
130 135 140
Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser
145 150 155 160
Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Val
165 170 175
Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser Val Asp His Ser
180 185 190
Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Asp Pro Thr Ile
195 200 205
Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn Leu Ser Leu Ser
210 215 220
Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile Asp
225 230 235 240
Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile Ser Asn Ile Thr
245 250 255
Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn Asn Ser Ala Ser
260 265 270
Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val Ser Ala Leu Ser
275 280 285
Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Thr
290 295 300
Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp Thr Gly
305 310 315 320
Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser Ser Glu
325 330 335
Arg Met Lys Leu Ser Gln Gly Asn Ile Thr Leu Ser Ile Asn Pro Val
340 345 350
Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn Pro Ile
355 360 365
Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr Asn Ala
370 375 380
Leu Pro Gln Glu Asn Leu Ile Asn Val Asp Gly Ser Gly Leu Asn Asp
385 390 395 400
Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Ala Arg Ala His His
405 410 415
His His His His
420
<210> 162
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> avi-标签
<400> 162
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15
Claims (27)
1.一种超激动性CD28抗原结合分子,其能够与CD28二价结合并且包含
(a)两个或更多个能够与CD28特异性结合的抗原结合结构域,
(b)至少一个能够与肿瘤相关抗原特异性结合的抗原结合结构域,和
(c)由能够稳定缔合的第一亚基和第二亚基构成的Fc结构域,其包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代。
2.根据权利要求1所述的超激动性CD28抗原结合分子,其中所述Fc结构域属于人IgG1亚类并且包含氨基酸突变L234A、L235A和P329G(根据Kabat EU索引编号)。
3.根据权利要求1或2所述的超激动性CD28抗原结合分子,其中所述能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(i)重链可变区(VHCD28),其包含SEQ ID NO:20的重链互补决定区CDR-H1、SEQ ID NO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的轻链互补决定区CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3;或
(ii)重链可变区(VHCD28),其包含SEQ ID NO:36的CDR-H1、SEQ ID NO:37的CDR-H2和SEQ ID NO:38的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:39的CDR-L1、SEQ IDNO:40的CDR-L2和SEQ ID NO:41的CDR-L3。
4.根据权利要求1至3中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含SEQID NO:20的CDR-H1、SEQ ID NO:21的CDR-H2和SEQ ID NO:22的CDR-H3;以及轻链可变区(VLCD28),其包含SEQ ID NO:23的CDR-L1、SEQ ID NO:24的CDR-L2和SEQ ID NO:25的CDR-L3。
5.根据权利要求1至4中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含与SEQID NO:26的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCD28),其包含与SEQ ID NO:27的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
6.根据权利要求1至3中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CD28特异性结合的抗原结合结构域中的每一个均包含:重链可变区(VHCD28),其包含选自由SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51组成的组的氨基酸序列;以及轻链可变区(VLCD28),其包含选自由SEQ ID NO:27、SEQ ID NO:52、SEQ ID NO:53、SEQID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQID NO:60和SEQ ID NO:61组成的组的氨基酸序列。
7.根据权利要求1至3或6中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CD28特异性结合的抗原结合结构域中的每一个均包含
(a)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(b)重链可变区(VHCD28),其包含SEQ ID NO:47的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(c)重链可变区(VHCD28),其包含SEQ ID NO:51的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:61的氨基酸序列,或
(d)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(e)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:54的氨基酸序列,或
(f)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(g)重链可变区(VHCD28),其包含SEQ ID NO:46的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(h)重链可变区(VHCD28),其包含SEQ ID NO:43的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列,或
(i)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:53的氨基酸序列,或
(j)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:59的氨基酸序列,或
(k)重链可变区(VHCD28),其包含SEQ ID NO:42的氨基酸序列,以及轻链可变区(VLCD28),其包含SEQ ID NO:27的氨基酸序列。
8.根据权利要求1至7中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CD28特异性结合的抗原结合结构域中的每一个均为Fab片段。
9.根据权利要求1至8中任一项所述的超激动性CD28抗原结合分子,其中所述能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与癌胚抗原(CEA)特异性结合的抗原结合结构域。
10.根据权利要求1至9中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含:(i)CDR-H1,其包含SEQ ID NO:127的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:128的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:129的氨基酸序列;以及轻链可变区(VLCEA),其包含:(iv)CDR-L1,其包含SEQ ID NO:130的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:131的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:132的氨基酸序列。
11.根据权利要求1至10中任一项所述的超激动性CD28抗原结合分子,其中所述能够与CEA特异性结合的抗原结合结构域包含:重链可变区(VHCEA),其包含与SEQ ID NO:133的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLCEA),其包含与SEQ ID NO:134的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
12.根据权利要求1至8中任一项所述的超激动性CD28抗原结合分子,其中所述能够与肿瘤相关抗原特异性结合的抗原结合结构域为能够与成纤维细胞活化蛋白(FAP)特异性结合的抗原结合结构域。
13.根据权利要求1至8或12中任一项所述的超激动性CD28抗原结合分子,其中所述能够与FAP特异性结合的抗原结合结构域包含
(a)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:12的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:13的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:14的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:15的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:16的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:17的氨基酸序列;或
(b)重链可变区(VHFAP),其包含:(i)CDR-H1,其包含SEQ ID NO:4的氨基酸序列,(ii)CDR-H2,其包含SEQ ID NO:5的氨基酸序列,和(iii)CDR-H3,其包含SEQ ID NO:6的氨基酸序列;以及轻链可变区(VLFAP),其包含:(iv)CDR-L1,其包含SEQ ID NO:7的氨基酸序列,(v)CDR-L2,其包含SEQ ID NO:8的氨基酸序列,和(vi)CDR-L3,其包含SEQ ID NO:9的氨基酸序列。
14.根据权利要求1至8或12或13中任一项所述的超激动性CD28抗原结合分子,其中所述能够与FAP特异性结合的抗原结合结构域包含
(a)重链可变区(VHFAP),其包含与SEQ ID NO:18的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ ID NO:19的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;或
(b)重链可变区(VHFAP),其包含与SEQ ID NO:10的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列;以及轻链可变区(VLFAP),其包含与SEQ ID NO:11的氨基酸序列至少约95%、96%、97%、98%、99%或100%相同的氨基酸序列。
15.根据权利要求1至14中任一项所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和所述包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的VH和VL结构域,其中所述VH结构域经由肽连接基与两条重链中的一条的C末端联结,并且其中所述VL结构域经由肽连接基与第二重链的C末端联结。
16.根据权利要求1至14中任一项所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和所述包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)能够与肿瘤相关抗原特异性结合的crossFab片段,其经由肽连接基与两条重链中的一条的C末端联结。
17.根据权利要求1至14中任一项所述的超激动性CD28抗原结合分子,其包含
(a)抗体的两条轻链和两条重链,其包含两个能够与CD28特异性结合的Fab片段和所述包含一个或多个降低所述抗原结合分子与Fc受体的结合亲和力和/或效应子功能的氨基酸取代的Fc结构域,以及
(b)两个能够与肿瘤相关抗原特异性结合的crossFab片段,其中一个crossFab片段经由肽连接基与两条重链中的一条的C末端联结,并且其中另一个crossFab片段经由肽连接基与第二重链的C末端联结。
18.一种多核苷酸,其编码根据段落1至17中任一项所述的双特异性抗原结合分子。
19.一种宿主细胞,其包含根据权利要求18所述的多核苷酸。
20.一种生产根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子的方法,所述方法包括在适于表达所述双特异性抗原结合分子的条件下培养根据权利要求19所述的宿主细胞。
21.一种药物组合物,其包含根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子以及至少一种药用赋形剂。
22.根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子或根据权利要求21所述的药物组合物,其用作药物。
23.根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子或根据权利要求21所述的药物组合物,其用于治疗癌症。
24.根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子,其用于治疗癌症,其中所述超激动性CD28抗原结合分子与化疗剂、放疗和/或用于癌症免疫疗法的其他药剂联合施用。
25.根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子或根据权利要求21所述的药物组合物在制造用于治疗癌症的药物中的用途。
26.一种抑制个体中肿瘤细胞生长的方法,所述方法包括向所述个体施用有效量的根据权利要求1至17中任一项所述的超激动性CD28抗原结合分子或根据权利要求22所述的药物组合物,以抑制所述肿瘤细胞的生长。
27.一种治疗癌症的方法,所述方法包括向所述个体施用治疗有效量的根据权利要求1至20中任一项所述的超激动性CD28抗原结合分子或根据权利要求24所述的药物组合物。
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WO2018177966A1 (en) * | 2017-03-27 | 2018-10-04 | F. Hoffmann-La Roche Ag | Improved antigen binding receptors |
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WO2023155845A1 (zh) * | 2022-02-16 | 2023-08-24 | 上海优替济生生物医药有限公司 | 人源化抗cd28抗体及其与抗cd40的双特异性抗体 |
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TW202030204A (zh) | 2020-08-16 |
US20200223925A1 (en) | 2020-07-16 |
AR117728A1 (es) | 2021-08-25 |
EP3898683A1 (en) | 2021-10-27 |
WO2020127628A1 (en) | 2020-06-25 |
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