CN110257338A - 嵌合细胞因子受体 - Google Patents
嵌合细胞因子受体 Download PDFInfo
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- CN110257338A CN110257338A CN201910541671.1A CN201910541671A CN110257338A CN 110257338 A CN110257338 A CN 110257338A CN 201910541671 A CN201910541671 A CN 201910541671A CN 110257338 A CN110257338 A CN 110257338A
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Abstract
实施方案涉及使用嵌合细胞因子受体(CCR)细胞治疗癌症的组合物和用途。一些实施方案涉及编码CCR的分离的核酸序列，其中CCR包含细胞外结构域，跨膜结构域和细胞内结构域，细胞外结构域结合免疫抑制剂，细胞内结构域传递细胞因子信号。一些实施方案涉及包含嵌合细胞因子受体(CCR)的修饰细胞，其中CCR包含细胞外结构域，跨膜结构域和胞内结构域，细胞外结构域结合免疫抑制剂，细胞内结构域传递细胞因子信号。
Description
技术领域
本公开涉及修饰的细胞，具体涉及使用嵌合细胞因子受体(CCR)的细胞治疗癌症的组合物和用途。
背景技术
使用系统性细胞因子可能起到抗肿瘤作用，但会带来全身副作用。因此，如何增强细胞因子在特定部位/肿瘤微环境中的功能变得更加重要。例如，表达PD-1的CAR-T细胞在肿瘤微环境中被PD-L1抑制，使得免疫疗法效率提高。
发明内容
本发明所采用的技术方案如下：
实施方案涉及使用嵌合细胞因子受体(CCR)细胞治疗癌症的组合物和用途。一些实施方案涉及编码嵌合细胞因子受体(CCR)的分离的核酸序列，其中CCR包含细胞外结构域，跨膜结构域和细胞内结构域，细胞外结构域结合免疫抑制剂，细胞内结构域传递细胞因子信号。一些实施方案涉及包含嵌合细胞因子受体(CCR)的修饰细胞，其中CCR包含细胞外结构域，跨膜结构域和胞内结构域，细胞外结构域结合免疫抑制剂，细胞内结构域传递细胞因子信号。
本发明内容并非旨在确定所要求保护的主题的关键特征或基本特征，也并非旨在用于限制所要求保护的主题的范围。
除非另有定义，本文所使用的所有技术和科学术语具有如本公开所属的领域的普通技术人员通常理解的相同的含义。虽然在本公开的实践或测试中可以使用与本文描述的方法和材料类似或等同的任何方法和材料，但是描述了优选的方法和材料。对于本公开的目的，下列术语定义如下。
本文使用冠词“一(a)”和“一个(an)”指的是一个或多于一个(即，指的是至少一个)该冠词的语法对象。举例来说，“元件”表示一个元件或多于一个元件。
所谓“约”是指数量，水平，数值，数量，频率，百分比，尺寸，大小，数量，重量或长度变化多达20,15,10,9,8,7,6,4,3,2或1％至参考数量，水平，数值，数量，频率，百分比，尺寸，大小，数量，重量或长度。
如本文所用，术语“活化”是指已经充分刺激以诱导可检测的细胞增殖的细胞的状态。激活还可以与诱导的细胞因子产生和可检测的效应子功能相关联。术语“活化的T细胞”特别指正在进行细胞分裂的T细胞。
术语“抗体”以最广泛的含义使用，并且是指单克隆抗体(包括全长单克隆抗体)，多克隆抗体，多特异性抗体(例如双特异性抗体)和抗体片段，只要它们表现出期望的生物学活性或功能。本公开中的抗体可以以各种形式存在，包括例如多克隆抗体，单克隆抗体，Fv，Fab和F(ab)2以及单链抗体和人源化抗体(Harlow等，1999，Antibodies：A LaboratoryManual，Cold Spring Harbor，New York；Houston等，1988，Proc.Natl.Acad.Sci.USA 85：5879-5883；Bird等，1988，Science 242：423-426)。
术语“抗体片段”是指全长抗体的一部分，例如抗体的抗原结合区或可变区。抗体片段的其他实例包括Fab，Fab'，F(ab')2和Fv片段；双体；线性抗体；单链抗体分子；和由抗体片段形成的多特异性抗体。
术语“Fv”是指含有完整抗原识别和结合位点的最小抗体片段。该片段由紧密非共价结合的一个重链和一个轻链可变区结构域的二聚体组成。从这两个结构域的折叠产生六个超变环(每个来自H和L链的3个环)，其贡献氨基酸残基用于抗原结合并赋予抗体结合抗原的特异性。然而，甚至单个可变结构域(或仅包含对抗原特异性的三个互补决定区(CDR)的Fv的一半)具有识别和结合抗原的能力，尽管其亲和力低于整个结合位点(二聚体)。
如本文所用，“抗体重链”是指存在于所有抗体分子中的天然存在的构象中的两种类型的多肽链中较大的一种。如本文所用，“抗体轻链”是指存在于其天然存在的构象中的所有抗体分子中的两种类型的多肽链中较小的一种。κ和λ轻链是指两种主要抗体轻链同种型。
术语“合成抗体”是指使用重组DNA技术产生的抗体，例如由噬菌体表达的抗体。该术语还包括通过合成编码抗体的DNA分子和表达DNA分子以获得抗体或获得编码抗体的氨基酸而产生的抗体。合成DNA是使用本领域已知的技术获得的。
术语“抗原”是指引起免疫应答的分子，其可以涉及抗体产生或特异性免疫活性细胞的活化或两者。抗原包括任何大分子，包括所有蛋白质或肽，或来源于重组或基因组DNA的分子。例如，包含编码引起免疫应答的蛋白质或肽的核苷酸序列或部分核苷酸序列的DNA，因此编码如本文所用的术语“抗原”。抗原不需要仅由基因的全长核苷酸序列编码。可以从包括组织样本，肿瘤样本，细胞或生物流体的生物样本产生，合成或衍生抗原。
如本文所用，术语“抗肿瘤作用”是指与肿瘤体积减少，肿瘤细胞数量减少，转移瘤数量减少，肿瘤细胞增殖减少，肿瘤细胞存活，具有肿瘤细胞的受试者的预期寿命增加，或与癌症相关的各种生理症状的改善。“抗肿瘤效应”也可以通过肽，多核苷酸，细胞和抗体首先预防肿瘤发生的能力来表现。
术语“自体抗原”指的是由免疫系统错误识别为外源的抗原。自体抗原包括细胞蛋白质、磷蛋白质、细胞表面蛋白质、细胞脂质、核酸、糖蛋白，包括细胞表面受体。
术语“自体”用于描述源自受试者的材料，随后将其重新引入相同的受试者。
术语“同种异体”用于描述来源于相同物种的不同受试者的移植物。作为例子，供体受试者可以是相关或不相关或受体受试者，但供体受试者具有与受体受试者相似的免疫系统标记。
术语“异种”用于描述来源于不同物种的受试者的移植物。举例来说，供体受试者来自与受体受试者不同的物种，并且供体受试者和受体受试者可以是遗传和免疫学不相容的。
术语“癌症”用于指代以异常细胞的快速且不受控制的生长为特征的疾病。癌细胞可以局部或通过血液和淋巴系统传播到身体的其他部位。各种癌症的例子包括乳腺癌，前列腺癌，卵巢癌，宫颈癌，皮肤癌，胰腺癌，结直肠癌，肾癌，肝癌，脑癌，淋巴瘤，白血病，肺癌等。
在整个本说明书中，除非上下文另有要求，词语“包含”和“包括”将被理解为指包括所陈述的步骤或元素或者步骤或元素的集合，但是不排除任何其它步骤或元素或者步骤或元素的集合。
短语“由...组成”意味着包括并且限于在短语“由...组成”之后的任何事物。因此，短语“由...组成”表示列出的元素是必需的或强制性的，并且不存在其它元素可以存在。
短语“基本上由......组成”意在包括在该短语之后列出的任何要素，并且可以包括不干扰或有助于本公开中所列举的要素所指定的活动或动作的其他要素。因此，短语“基本上由......组成”表示列出的要素是必需的或强制性的，但是其他要素是可选的并且可能存在或可能不存在，这取决于它们是否影响所列要素的活性或作用。
术语“互补的”和“互补”指的是通过碱基配对原则相关的多核苷酸(即核苷酸的序列)。例如，序列“A-G-T”与序列“T-C-A”是互补的。互补可以是“部分的”，其中仅一些核酸的碱基根据碱基配对原则进行匹配。或者，核酸之间可存在“完全”或“总体”的互补。核酸链之间的互补程度对核酸链之间杂交的效率和强度具有显著影响。
术语“对应于”或“对应于”是指(a)具有与参考多核苷酸序列的全部或一部分基本上相同或互补或编码与氨基酸序列相同的氨基酸序列的核苷酸序列的多核苷酸在肽或蛋白质中；或(b)具有与参考肽或蛋白质中的氨基酸序列基本上相同的氨基酸序列的肽或多肽。
术语“共刺激配体”是指特异性结合T细胞上的同源共刺激分子的抗原呈递细胞(例如，APC，树突细胞，B细胞等)上的分子，由此提供除了通过例如TCR/CD3复合物与负载有肽的MHC分子的结合所提供的初级信号之外，信号还介导包括增殖，活化，分化和其他中的至少一种的T细胞应答细胞反应。共刺激配体可以包括B7-1(CD80)，B7-2(CD86)，PD-L1，PD-L2,4-1BBL，OX40L，诱导型共刺激配体(ICOS-L)，细胞间粘附分子ICAM)，CD30L，CD40，CD70，CD83，HLA-G，MICA，MICB，HVEM，淋巴毒素β受体，3/TR6，ILT3，ILT4，HVEM，CD7的配体，与Toll配体受体结合的激动剂或抗体和与B7-H3特异性结合的配体。共刺激配体还尤其包括特异性结合存在于T细胞上的共刺激分子如CD27，CD28,4-1BB，OX40，CD30，CD40，PD-1的激动剂或抗体，ICOS，淋巴细胞功能相关抗原-1(LFA-1)，CD2，CD7，LIGHT，NKG2C，B7-H3和特异性结合CD83的配体。
术语“共刺激分子”是指T细胞上的同源结合配偶体，其与共刺激配体特异性结合，由此介导T细胞的共刺激反应，例如增殖。共刺激分子包括MHC I类分子，BTLA和Toll样受体。
术语“共刺激信号”是指与主要信号例如TCR/CD3连接结合的信号导致T细胞增殖和/或关键分子的上调或下调。术语“疾病”和“条件”可以互换使用，也可以不同，因为特定的疾病或病症可能没有已知的病原体(因此病因学还没有制定出来)，因此它尚未被认为是一种疾病，而只是作为一种疾病不良状况或综合征，其中临床医师已经确定了或多或少的特定症状。术语“疾病”是受试者的健康状况，其中受试者不能维持体内平衡，并且其中如果疾病未得到改善，则受试者的健康持续恶化。相反，受试者中的“障碍”是动物能够维持动态平衡的健康状态，但其中动物的健康状况不如没有障碍时那样有利。如果不进行治疗，病症不一定会导致动物健康状况的进一步下降。
术语“有效”是指足以实现期望的，预期的或预期的结果。例如，治疗背景下的“有效量”可以是足以产生治疗或预防益处的化合物的量。
术语“编码”是指多核苷酸(例如基因，cDNA或mRNA)中特定核苷酸序列的固有性质，用作在生物过程中合成其他聚合物和大分子的模板，其具有确定的序列核苷酸(即，rRNA，tRNA和mRNA)或确定的氨基酸序列以及由此产生的生物学性质。因此，如果对应于该基因的mRNA的转录和翻译在细胞或其他生物系统中产生蛋白质，则该基因编码蛋白质。编码链的核苷酸序列与mRNA序列相同(除了“T”被“U”代替外)并且通常在序列表中提供，非编码链用作模板对于基因或cDNA的转录而言，可以称为编码该基因或cDNA的蛋白质或其他产物。
术语“外源性”是指在野生型细胞或生物中不天然存在但通常通过分子生物学技术引入细胞的分子。外源多核苷酸的实例包括编码所需蛋白质的载体，质粒和/或人造核酸构建体。关于多核苷酸和蛋白质，术语“内源性”或“天然的”是指可以在给定的野生型细胞或生物体中发现的天然存在的多核苷酸或氨基酸序列。而且，从第一生物体中分离并通过分子生物学技术转移至第二生物体的特定多核苷酸序列通常被认为是关于第二生物体的“外源”多核苷酸或氨基酸序列。在具体的实施方案中，可以通过分子生物学技术将多核苷酸序列“引入”已经含有这种多核苷酸序列的微生物中，例如以产生另外天然存在的多核苷酸序列的一个或多个另外的拷贝，并且由此有助于过表达编码的多肽。
术语“表达”是指由其启动子驱动的特定核苷酸序列的转录和/或翻译。
术语“表达载体”是指包含重组多核苷酸的载体，所述重组多核苷酸包含与待表达的核苷酸序列可操作地连接的表达控制序列。表达载体包括足够的用于表达的顺式作用元件；用于表达的其他元件可以由宿主细胞提供或在体外表达系统中提供。表达载体包括本领域已知的所有那些，如掺入重组多核苷酸的粘粒，质粒(例如裸露的或包含在脂质体中)和病毒(例如慢病毒，逆转录病毒，腺病毒和腺伴随病毒)。
术语“同源的”是指当两个比较的序列中的两个位置被相同的碱基或氨基酸单体亚基占据时，例如如果两个多肽DNA分子被腺嘌呤占据，那么分子在该位置是同源的。两个序列之间的同源性百分比是由两个序列共有的匹配或同源位置的数量除以比较的位置数×100的函数。例如，如果两个序列中10个位置中的6个匹配或同源，那么这两个序列是60％同源的。举例来说，DNA序列ATTGCC和TATGGC具有50％的同源性。当两个序列比对以给出最大的同源性时进行比较。
术语“免疫球蛋白”或“Ig”指的是起到抗体作用的一类蛋白质。包括在该类蛋白质中的五个成员为IgA、IgG、IgM、IgD和IgE。IgA为存在于身体分泌物(诸如唾液、泪液、母乳)、胃肠分泌物以及呼吸道和泌尿生殖道的粘液分泌物中的初级抗体。IgG是最常见的循环抗体。IgM是在多数受试者的初级免疫应答中产生的主要免疫球蛋白。它是在凝集、补体结合和其它抗体应答中最有效的免疫球蛋白，并且在抵御细菌和病毒方面是很重要的。IgD是不具有已知抗体功能但可用作抗原受体的免疫球蛋白。IgE是在暴露于过敏原后，通过引起从肥大细胞和嗜碱性粒细胞释放介体而介导速发型超敏反应的免疫球蛋白。
术语“分离的”是指基本上或基本上不含通常伴随其天然状态的组分的材料。该材料可以是细胞或大分子，如蛋白质或核酸。例如，如本文所用的“分离的多核苷酸”是指已经从天然存在状态的侧翼序列中纯化的多核苷酸，例如已经从通常是正常序列中去除的DNA片段与片段相邻。或者，本文所用的“分离的肽”或“分离的多肽”等是指肽或多肽分子从其天然细胞环境的体外分离和/或纯化，以及与其他组分细胞。
术语“基本上纯化的”是指基本上来自通常与其天然状态相关的组分的材料。例如，基本上纯化的细胞是指与其天然存在或天然状态下正常结合的其他细胞类型分开的细胞。在一些情况下，基本上纯化的细胞群指的是均质细胞群。在其他情况下，这个术语简单地指的是与它们天然状态下与其天然相关的细胞分开的细胞。在一些实施方案中，细胞在体外培养。在其他实施方案中，细胞不在体外培养。
在本公开的上下文中，使用下列普遍存在的核酸碱基的缩写。“A”指的是腺苷，“C”指的是胞嘧啶，“G”指的是鸟苷，“T”指的是胸苷，以及“U”指的是尿苷。
除非另有说明，“编码氨基酸序列的核苷酸序列”包括彼此互为简并版本并且编码相同的氨基酸序列的所有核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可包括内含子，在一定程度上编码蛋白质的核苷酸序列可在某些版本中包含(一个或多个)内含子。
术语“慢病毒”是指逆转录病毒科的一个属。慢病毒在能够感染非分裂细胞的逆转录病毒中是独特的；它们可以将大量的遗传信息输送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV，SIV和FIV都是慢病毒的例子。来自慢病毒的载体提供了实现显着水平的体内基因转移的手段。
术语“调节”是指与不存在治疗或化合物的受试者中的反应水平相比，调节受试者中反应水平的可检测增加或减少，和/或与在其他方面相同但未经处理的主题中的回应。该术语包括扰乱和/或影响天然信号或应答，由此介导受试者，优选人的有益治疗反应。
当核酸置于与另一核酸序列的功能关系中时，它是“可操作地连接”的。例如，如果将前序列或分泌前导序列的DNA表达为参与多肽分泌的前蛋白，则其与多肽的DNA可操作地连接；如果启动子或增强子影响序列的转录，则其与编码序列可操作地连接；或者如果核糖体结合位置被定位以便于翻译，则该核酸体结合位点与编码序列可操作地连接。
术语“在转录控制下”是指与多核苷酸有效连接并处于正确位置和方向的启动子，以控制RNA聚合酶的转录起始和多核苷酸的表达。
术语“过度表达”的肿瘤抗原或肿瘤抗原的“过表达”旨在表示肿瘤抗原在来自疾病区域的细胞中的异常表达水平，所述疾病区域例如患者相关的特定组织或器官内的实体肿瘤到来自该组织或器官的正常细胞中的表达水平。可以通过本领域已知的标准测定来确定具有肿瘤抗原过表达的以实体瘤或血液恶性肿瘤为特征的患者。
组合物的术语“肠胃外施用”包括例如皮下(sc)，静脉内(iv)，肌内(im)，胸骨内注射或输注技术。
术语“患者”，“受试者”和“个体”等在本文中可互换使用，并且指适用于本文所述方法的任何人类，动物或活的生物体。在某些非限制性实施方案中，患者，受试者或个体是人或动物。在一些实施方案中，术语“受试者”旨在包括其中可引发免疫应答的活生物体(例如，哺乳动物)。受试者的实例包括人，以及动物如狗，猫，小鼠，大鼠及其转基因物种。
需要治疗或需要治疗的受试者包括具有需要治疗的疾病，病症或障碍的受试者。有需要的受试者还包括需要治疗以预防疾病，病症或障碍的受试者。
术语“多核苷酸”或“核酸”是指mRNA，RNA，cRNA，rRNA，cDNA或DNA。该术语通常是指至少10个碱基长度的核苷酸的聚合形式，或者是核糖核苷酸或者脱氧核苷酸，或者是任一种核苷酸的修饰形式。该术语包括所有形式的核酸，包括单链和双链形式的核酸。
术语“多核苷酸变体”和“变体”等是指与参考多核苷酸序列显示出实质序列同一性的多核苷酸或在下文定义的严格条件下与参考序列杂交的多核苷酸。这些术语还包括通过添加，缺失或取代至少一个核苷酸而与参考多核苷酸不同的多核苷酸。因此，术语“多核苷酸变体”和“变体”包括其中一个或多个核苷酸已被添加或缺失或被不同核苷酸置换的多核苷酸。就这一点而言，本领域众所周知的是，可以对参照多核苷酸进行包括突变，添加，缺失和取代的某些改变，由此改变的多核苷酸保留参照多核苷酸的生物学功能或活性或具有与参考多核苷酸的关系(即优化的)。多核苷酸变体包括例如与参考多核苷酸序列具有至少50％(以及至少51％至至少99％以及所有整数百分比之间，例如90％，95％或98％)序列同一性的多核苷酸在此描述。术语“多核苷酸变体”和“变体”还包括天然存在的等位基因变体和直向同源物。
术语“多肽”，“多肽片段”，“肽”和“蛋白质”在本文中可互换使用，是指氨基酸残基的聚合物以及其变体和合成类似物。因此，这些术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是合成的非天然存在的氨基酸，例如相应天然存在的氨基酸的化学类似物，以及天然存在的氨基酸聚合物。在某些方面，多肽可以包括通常催化(即增加各种化学反应的速率)的酶促多肽或“酶”。
术语“多肽变体”是指通过添加，缺失或取代至少一个氨基酸残基而与参考多肽序列区分的多肽。在某些实施方案中，通过一个或多个取代将多肽变体与参考多肽区分开来，所述取代可以是保守的或非保守的。在某些实施方案中，多肽变体包含保守取代，并且就此而言，本领域中熟知可以将一些氨基酸改变为具有广泛相似性质的氨基酸，而不改变多肽活性的性质。多肽变体还包括其中一个或多个氨基酸已被添加或缺失或被不同氨基酸残基置换的多肽。
术语“启动子”是指由启动多核苷酸序列特异性转录所需的细胞合成机器或引入的合成机器识别的DNA序列。术语“表达控制序列”是指在特定宿主生物中表达可操作连接的编码序列所必需的DNA序列。例如，适用于原核生物的控制序列包括启动子，任选的操纵子序列和核糖体结合位点。已知真核细胞利用启动子，聚腺苷酸化信号和增强子。
术语“结合”，“结合”或“与......相互作用”是指识别并粘附于样品或生物体中特定第二分子的分子，但基本上不识别或粘附于样品中其他结构不相关的分子。如本文关于抗体所使用的术语“特异性结合”是指识别特异性抗原但实质上不识别或结合样品中其他分子的抗体。例如，特异性结合来自一个物种的抗原的抗体也可以结合来自一个或多个物种的抗原。但是，这种跨物种反应性本身并不改变抗体的特异性分类。在另一个实例中，特异性结合抗原的抗体也可以结合不同的抗原等位基因形式。然而，这种交叉反应本身并不改变抗体的特异性分类。在一些情况下，术语“特异性结合”或“特异性结合”可用于指抗体，蛋白质或肽与第二化学物质的相互作用，意指相互作用取决于存在化学物种上的特定结构(例如抗原决定簇或表位)；例如，抗体识别并结合特定蛋白质结构而不是任何蛋白质。如果抗体对表位“A”具有特异性，则在含有标记的“A”和抗体的反应中含有表位A的分子(或游离的，未标记的A)的存在将减少与抗体结合的标记的A的量。
“统计学上显著的”是指结果不太可能是偶然发生的。统计显著性可通过本领域已知的任何方法来确定。通常使用的显著性测量值包括P值，它是在无效假设是真的情况下所观察到的事件将发生的频率或概率。如果获得的p值小于显著性水平，则无效假设被拒绝。在简单的情况下，显著性水平被限定在0.05或更小的p值。“下降的”或“减少的”或“更少的”量通常是“统计学上显著的”或生理学上显著的量，并且可以包括是本文中描述的量或水平的约1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2、2.5、3、3.5、4、4.5、5、6、7、8、9、10、15、20、30、40或50倍或更多倍(例如，100、500、1000倍)(包括在之间和在1以上的所有整数和小数点，例如，1.5、1.6、1.7、1.8等)的下降。
术语“刺激”是指由刺激分子(例如TCR/CD3复合物)与其同源配体结合而诱导的初级应答，由此介导信号转导事件，例如经由TCR/CD3复合物的信号转导。刺激可以介导某些分子的改变的表达，例如TGF-β的下调和/或细胞骨架结构的重组。
术语“刺激分子”是指特异性结合存在于抗原呈递细胞上的同源刺激配体的T细胞上的分子。例如，源自刺激分子的功能性信号传导结构域是与T细胞受体复合物相关的ζ链。
术语“刺激性配体”是指当存在于抗原呈递细胞(例如，APC，树突细胞，B细胞等)上时可以与同源结合配偶体特异性结合的配体(在本文中称为作为“刺激分子”)在细胞(例如T细胞)上的表达，从而介导T细胞的主要应答，包括激活，启动免疫应答，增殖和类似过程。刺激性配体在本领域中是公知的，并且尤其包括装载有肽，抗CD3抗体，超级激动剂抗CD28抗体和超级激动剂抗CD2抗体的MHC I类分子。
术语“治疗剂”是指治疗和/或预防。治疗效果通过抑制，缓解或根除疾病状态或减轻疾病状态症状而获得。
术语“治疗有效量”是指将引起研究人员，兽医，医生或另一临床医生正在寻求的组织，系统或受试者的生物或医学反应的主题化合物的量。术语“治疗有效量”包括当施用时足以预防所治疗的病症或疾病的一种或多种症状或症状的发展或在一定程度上减轻所述病症或疾病的一种或多种症状或症状的量的化合物。治疗有效量将取决于待治疗的受试者的化合物，疾病及其严重程度和年龄，体重等而变化。
术语“治疗疾病”是指降低受试者经历的疾病或病症的至少一种体征或症状的频率或严重程度。
术语“转染的”或“转化的”或“转导的”是指将外源核酸转移或引入宿主细胞的过程。“转染的”或“转化的”或“转导的”细胞是已经用外源核酸转染，转化或转导的细胞。细胞包括主要受试细胞及其后代。
术语“载体”是指包含分离的核酸并且可用于将分离的核酸递送至细胞内部的多核苷酸。本领域已知许多载体，包括线性多核苷酸，与离子或两亲化合物相关的多核苷酸，质粒和病毒。因此，术语“载体”包括自主复制质粒或病毒。该术语还包括便于将核酸转移入细胞的非质粒和非病毒化合物，例如多聚赖氨酸化合物，脂质体等。病毒载体的实例包括腺病毒载体，腺伴随病毒载体，逆转录病毒载体等。例如，慢病毒是复杂的逆转录病毒，除常见的逆转录病毒基因gag，pol和env外，还含有其他具有调控或结构功能的基因。慢病毒载体在本领域中是众所周知的。慢病毒的一些例子包括人免疫缺陷病毒：HIV-1，HIV-2和猿猴免疫缺陷病毒：SIV。通过多重减毒HIV毒力基因产生慢病毒载体，例如，缺失基因env，vif，vpr，vpu和nef使载体生物安全。
范围：在整个本公开中，本公开的各个方面可以以范围格式给出。应当理解，范围格式的描述仅是为了方便和简洁，并且不应当被解释为对本公开的范围的硬性限制。因此，范围的描述应被认为具有具体公开的所有可能的子范围以及处于那个范围内的单个数值。例如，范围的描述诸如从1至6应被认为具有具体公开的子范围诸如从1至3、从1至4、从1至5、从2至4、从2至6、从3至6等，以及那个范围内的单个数值，例如，1、2、2.7、3、4、5、5.3和6。不管范围的宽度如何，这一点都是适用的。
本公开的实施方案利用炎性因子受体来逆转CAR T细胞上肿瘤微环境的免疫抑制，并将逆转与炎症因子相关联以增强CAR T细胞在肿瘤微环境中的功能。此外，在一些实施方案中，免疫抑制剂及其受体的亲和力可以增强。常规技术在CAR T细胞中过度表达炎性细胞因子。虽然单方面增加肿瘤微环境中炎症因子的浓度，但这些技术可能不能同时逆转CAR-T细胞的免疫抑制并增加CAR T细胞对炎性因子的调节水平。
一些实施方案涉及可以过表达CCR的CAR T细胞，并且可以将抑制信号转化为炎性因子的激活信号以增强CAR-T细胞的功能。在一些实施方案中，可以进行抑制性受体的细胞外结构域中的氨基酸取代以增强它们的受体-配体亲和力并获得与内源性受体竞争配体的优势。例如，CCR可以包括抑制性受体细胞外结构域，跨膜区和内部炎性因子受体的细胞内结构域。
一些实施方案涉及编码嵌合细胞因子受体(CCR)的分离的核酸序列，其中CCR包含细胞外结构域，跨膜结构域和细胞内结构域，细胞外结构域结合免疫抑制剂，细胞内结构域传递细胞因子信号。一些实施方案涉及包含分离的核酸序列的CAR细胞群体。一些实施方案涉及包含CAR细胞群的药物组合物。一些实施方案涉及在有需要的受试者中引起T细胞应答和/或治疗受试者的肿瘤的方法，所述方法包括向受试者施用有效量的组合物。例如，免疫抑制剂是指阻断或减少免疫应答的分子。
如本文所用的术语“嵌合细胞因子受体”是指包含来自一个受体的细胞因子结合部分与来自不同受体的细胞内信号传导部分相连的工程化受体。如本文所用的术语“细胞因子结合外结构域”是指细胞表面上与细胞因子结合的细胞因子受体部分。
一些实施方案涉及包含嵌合细胞因子受体(CCR)的修饰细胞，其中CCR包含细胞外结构域，跨膜结构域和胞内结构域，细胞外结构域结合免疫抑制剂，细胞内结构域传递细胞因子信号。
在一些实施方案中，免疫抑制剂包含检查点抑制剂。在一些实施方案中，检查点抑制剂是特异性结合免疫关卡蛋白的蛋白或多肽。在一些实施方案中，免疫关卡蛋白选自由CTLA4，PD-1，PD-L1，PD-L2，A2AR，B7-H3，B7-H4，BTLA，KIR，LAG3，TIM-3或VISTA。在一些实施方案中，多肽或蛋白质是抗体或其抗原结合片段。在一些实施方案中，检查点抑制剂是干扰性核酸分子。在一些实施方案中，干扰核酸分子是siRNA分子，shRNA分子或反义RNA分子。在一些实施方案中，检查点抑制剂选自nivolumab，pembrolizumab，pidilizumab，AMP-224，AMP-514，STI-A1110，TSR-042，RG-7446，BMS-936559，BMS-936558，MK-3475，CT O11，MPDL3280A，MEDI-4736，MSB-0020718C，AUR-012和STI-A1010。
在一些实施方案中，免疫蛋白包含PD-1，CTLA-4，LAG-3，TIM-3或BLTA。在一些实施方案中，与广泛类型相比，PD-1，CTLA-4，LAG-3，TIM-3或BLTA的胞外结构域被修饰以具有增强的受体-配体亲和力。在一些实施方案中，细胞内结构域包含IL-12R，IL-6R，IL-2R或CD27的信号转导细胞因子胞内结构域。
在一些实施方案中，CCR是免疫抑制剂受体的显性失活变体，或者分离的核酸能够向宿主细胞提供免疫抑制剂受体的显性失活变体。在一些实施方案中，CCR的细胞外结构域包含SEQ ID NO：45或47的氨基酸序列，CCD的跨膜结构域包含SEQ ID NO：49或51的氨基酸序列，和/或CCR的胞内结构域包含SEQ ID NO：53或55的氨基酸序列。显性失活突变具有改变的基因产物，其对野生型等位基因起拮抗作用。这些突变通常导致分子功能改变(通常无效)，并且以显性或半显性表型为特征。
在一些实施方案中，修饰的细胞包含嵌合抗原受体(CAR)或分离的核酸序列包含编码CAR的核酸序列。在一些实施方案中，CAR包含细胞外结构域，跨膜结构域和细胞内结构域，细胞外结构域结合抗原。在一些实施方案中，细胞内结构域包含共刺激信号传导区域，所述共刺激信号传导区域包含选自CD27，CD28,4-1BB，OX40，CD30，CD40，PD-1，ICOS，淋巴细胞功能-相关抗原-1(LFA-1)，CD2，CD7，LIGHT，NKG2C，B7-H3及其任何组合。在一些实施方案中，表皮生长因子受体(EGFR)，表皮生长因子受体(EGFRvIII)的变体III，人表皮生长因子受体2(HER2)，间皮素(MSLN)，前列腺特异性膜抗原(PSMA)，癌胚抗原(CEA)，二糖神经节苷脂2(GD2)，白介素-13Ra2(IL13Rα2)，磷脂酰肌醇蛋白聚糖-3(GPC3)，碳酸酐酶IX(CAIX)，L1细胞粘附分子(L1-CAM)，癌抗原125(CA125)(CD133)，成纤维细胞活化蛋白(FAP)，癌/睾丸抗原1B(CTAG1B)，粘蛋白1(MUC1)，叶酸受体-α(FR-α)，CD19，FZD10，TSHR，PRLR，Muc17，GUCY2C，CD207，CD3，CD5，B细胞成熟抗原(BCMA)或CD4。
在一些实施方案中，修饰的TCR源自患者中自发发生的肿瘤特异性T细胞。在一些实施方案中，修饰的TCR结合肿瘤抗原。在一些实施方案中，肿瘤抗原包含CEA，gp100，MART-1，p53，MAGE-A3或NY-ESO-1。在一些实施方案中，修饰的TCR包含TCRγ和TCRδ链或TCRα和TCRβ链。在一些实施方案中，可以分离表达对靶抗原具有高亲和力的TCR的T细胞克隆。在某些实施方案中，肿瘤浸润淋巴细胞(TIL)或外周血单核细胞(PBMC)可以在抗原呈递细胞(APC)存在下培养，所述抗原呈递细胞(APC)用表示已知在表达时引发显性T细胞应答的表位在确定的HLA等位基因的情况下。然后可以基于MHC-肽四聚体染色和/或识别和裂解用低滴定浓度的同源肽抗原脉冲的靶细胞的能力来选择高亲和力克隆。选择克隆后，通过分子克隆鉴定和分离TCRα和TCRβ链或TCRγ和TCRδ链。例如，对于TCRα和TCRβ链，然后使用TCRα和TCRβ基因序列产生理想地促进人T细胞中两种TCR链稳定，高水平表达的表达构建体。然后可以产生转导载体(例如γ逆转录病毒或慢病毒)并测试功能性(抗原特异性和功能亲合力)并用于产生临床批量的载体。然后使用等分试样的最终产物转导目标T细胞群体(通常从患者PBMC中纯化)，其在输注到患者体内之前被扩增。
可以实施各种方法来获得编码肿瘤反应性TCR的基因。Kershaw等人在文献中(Clin Transl Immunology.2014May 16；3(5):e16)提供了更多信息。在一些实施方案中，特定的TCR可以源自患者中自发发生的肿瘤特异性T细胞。此类抗原包括黑素细胞分化抗原MART-1和gp100，以及MAGE抗原和NY-ESO-1，在更广泛的癌症中表达。只要病毒蛋白由转化细胞表达，也可以分离针对病毒相关恶性肿瘤特异性的TCR。这一类别的恶性肿瘤包括肝癌和宫颈癌，与肝炎和乳头状瘤病毒以及Epstein-Barr病毒相关的恶性肿瘤有关。在一些实施方案中，TCR的靶抗原可包括CEA(例如，用于结肠直肠癌)，gp100，MART-1，p53(例如用于黑素瘤)，MAGE-A3(例如黑素瘤，食管和滑膜肉瘤)ESO-1(例如，用于黑素瘤和肉瘤以及多发性骨髓瘤)。
本公开的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的量和频率将由以下因素确定，诸如患者的病症、以及患者疾病的类型和严重度，尽管适当的剂量可由临床试验确定。
当指示“免疫有效量”，“抗肿瘤有效量”，“肿瘤抑制有效量”或“治疗量”时，可以确定要给予的本公开的组合物的精确量由医生考虑年龄，体重，肿瘤大小，感染或转移程度和患者(受试者)的状况的个体差异。可以说包含本文描述的T细胞的药物组合物可以以104至109细胞/kg体重，优选105至106细胞/kg体重的剂量给予，包括在这些范围内的所有整数值。T细胞组合物也可以在这些剂量下多次给药。细胞可以通过使用在免疫疗法中通常已知的输注技术来施用(参见例如Rosenberg等人，New Eng.J.of Med.319:1676,1988)。对于特定患者的最佳剂量和治疗方案可容易地由医学领域的技术人员通过监测患者的疾病迹象并相应地调整治疗来确定。在某些实施方案中，可能希望将活化的T细胞给予受试者，然后重新绘制血液(或进行单采)，收集活化和扩增的T细胞，并用这些活化和扩增的T细胞再灌注患者。这个过程可以每隔几周进行多次。在某些实施方案中，T细胞可以从10cc至400cc的抽血活化。在某些实施方案中，T细胞从20cc，30cc，40cc，50cc，60cc，70cc，80cc，90cc或100cc的抽血活化。不受理论束缚，使用这种多次抽血/多次再输注方案，可以选择某些T细胞群体。
本文所述药物组合物的施用可以以任何方便的方式进行，包括通过气雾吸入，注射，摄入，输血，植入或移植。本文所述的组合物可以皮下，皮内，瘤内，结节内，髓内，肌内，静脉内(iv)注射或腹膜内给予患者。在一些实施方案中，本公开内容的T细胞组合物通过皮内或皮下注射给予患者。在另一个实施方案中，本公开的T细胞组合物优选通过静脉内注射施用。T细胞的组合物可以直接注射到肿瘤，淋巴结或感染部位。在本公开的某些实施方案中，使用本文所述的方法或本领域已知的其中T细胞扩展至治疗水平的方法将活化和扩增的细胞与(例如，之前，同时或之后)任何数量的相关治疗方式，包括但不限于用抗病毒疗法，西多福韦和白细胞介素-2，阿糖胞苷(也称为ARA-C)等药物治疗或MS患者的那他珠单抗治疗或牛皮癣患者或其他患者的依法珠单抗治疗治疗PML患者。在进一步的实施方案中，本公开的T细胞可以与化学疗法，放射疗法，免疫抑制剂例如环孢菌素，硫唑嘌呤，甲氨蝶呤，霉酚酸酯和FK506，抗体或其他免疫清除剂例如CAM PATH，CD3抗体或其他抗体疗法，细胞毒素，氟达利宾，环孢菌素，FK506，雷帕霉素，麦考酚酸，类固醇，FR901228，细胞因子和辐射。这些药物抑制钙依赖性磷酸酶钙调磷酸酶(环孢菌素和FK506)或抑制对生长因子诱导的信号传导(雷帕霉素)重要的p70S6激酶。(Liu等，Cell 66:807-815,1991；Henderson等，Immun73:316-321,1991；Bierer等，Curr.Opin.Immun 5:763-773,1993)。在一些实施方案中，将本公开的细胞组合物与骨髓移植联合(例如，之前，同时或之后)，使用化疗剂如氟达拉滨的T细胞消融治疗，外部束放射疗法(XRT)，环磷酰胺或抗体如OKT3或CAMPATH。在其他实施方案中，本公开的细胞组合物在B细胞消融疗法例如与CD20反应的试剂例如Rituxan之后施用。例如，在一些实施方案中，受试者可接受高剂量化学疗法的标准治疗，然后进行外周血干细胞移植。在某些实施方案中，在移植后，受试者接受本公开的扩增的免疫细胞的输注。在其他实施方案中，扩张细胞在手术之前或之后施用。
待给予患者的上述治疗剂量将随着被治疗病症和治疗接受者的确切性质而变化。根据各种因素，医师可根据本领域公认的实践进行人用药剂量的缩放。
在美国专利No.5,315,418中提供了关于使用工程化或修饰的T细胞的癌症治疗方法的其他信息。美国专利US8,906,682，通过引用整体并入。
一些实施方案涉及制备修饰细胞的体外方法。该方法可以包括从受试者获得细胞的样品。例如，样品可以包括T细胞或T细胞祖细胞。该方法可进一步包括用编码至少CAR的DNA转染细胞，在选择性增强CAR表达T细胞增殖的培养基中离体培养CAR细胞群。
在一些实施方案中，样品是冷冻保存的样品。在一些实施方案中，细胞样品来自脐带血或来自受试者的外周血液样品。在一些实施方案中，细胞样品通过单采血液成分或静脉穿刺获得。在一些实施方案中，细胞样品是T细胞的亚群。
通过参考以下示例性实施例和示例来进一步描述本公开。提供这些示例性实施例和示例仅用于说明的目的，并非旨在限制，除非另有说明。因此，本公开绝不应被解释为限于以下示例性实施例和示例，而是应被解释为包含由于本文提供的教导而变得明显的任何和所有变化。
进一步，本文描述的实施方案涉及组合物在有此需要的受试者中引起T细胞应答和/或治疗受试者的肿瘤的用途，该方法包括向受试者施用有效量的组合物，其中所述组合物包含上述的修饰细胞。通过参考以下示例性实施例和示例来进一步描述本公开。提供这些示例性实施例和示例仅用于说明的目的，并非旨在限制，除非另有说明。因此，本公开绝不应被解释为限于以下示例性实施例和示例，而是应被解释为包含由于本文提供的教导而变得明显的任何和所有变化。
附图说明
参照附图描述具体实施方式。在不同附图中使用相同的参考数字表示类似或相同的项目。
图1是显示CAR和编码CAR和CCR的代表性质粒的结构示意图。
图2是显示CAR和在淋巴细胞上表达的CAR和CCR的示意图。
图3是显示在T细胞上表达的修饰的TCR和CCR的示意图。
图4是CCR的结构示意图，其中包含细胞外结构域，质膜或跨膜结构域和细胞内结构域。细胞外结构域的例子包括PD-1，CTLA-4，TIM-3和LAG-3的胞外结构域。跨膜结构域的例子包括PD-1，CTLA-4，TIM-3，LAG-3，CD8，CD28，IL-12R和IL-6R的跨膜结构域。细胞内结构域的例子包括IL-12R，IL-6R，IL-2R和CD27的细胞内结构域。在一些实施方案中，细胞外结构域的部分氨基酸可以被取代以增强配体/抗原与其受体之间的亲和力。
图5显示了用于在淋巴细胞(T细胞)上表达CAR和CCR的核酸载体的结构。
图6是已构建的嵌合趋化因子受体(CCRs)载体列表。
图7-10显示各载体的PD1和CAR在293T细胞中都是呈双阳性表达的。
图11-14显示嵌合趋化因子受体(CCRs)载体在T细胞上的表达。
图15显示CAR-CCR T细胞和Nalm6或Nalm6-PDL1细胞共培养的肿瘤细胞数量曲线。
图16显示CAR-CCR T细胞和Nalm6-PDL1细胞共培养7天的肿瘤细胞数量曲线。
图17-22显示CAR-CCR T细胞和Nalm6/Nalm6-PDL1细胞共培养24h的CD137表达。
图23和24显示CAR-CCR T细胞和Nalm6/Nalm6-PDL1细胞共培养24/48h的细胞因子释放水平。
具体实施方式
以下是示例性实施例：
1.编码嵌合细胞因子受体(CCR)的分离的核酸序列，其中所述CCR包含细胞外结构域，跨膜结构域和细胞内结构域，所述细胞外结构域结合免疫抑制剂，并且所述细胞内结构域传递细胞因子信号。
2.一种包含嵌合细胞因子受体(CCR)的修饰细胞，其中所述CCR包含细胞外结构域，跨膜结构域和细胞内结构域，所述细胞外结构域结合免疫抑制剂，并且所述细胞内结构域传递细胞因子信号。
3.根据实施例1-2中任一项所述的分离的核酸序列或修饰的细胞，其中所述免疫抑制剂包含检查点抑制剂。
4.根据实施例1-2中任一项所述的分离的核酸序列或修饰的细胞，其中所述免疫抑制剂是PD-1，CTLA-4，LAG-3，TIM-3或BLTA。
5.根据实施例4所述的分离的核酸序列或修饰的细胞，其中PD-1，CTLA-4，LAG-3，TIM-3或BLTA的胞外结构域被修饰以具有增强的受体-配体亲和力相比较于野生型。
6.根据实施例1-2中任一项所述的分离的核酸序列或修饰的细胞，其中所述细胞内结构域包含IL-12R，IL-6R，IL-2R或CD27的信号转导细胞因子胞内结构域。
7.根据实施例1-2中任一项所述的分离的核酸序列或修饰的细胞，其中所述CCR是所述免疫抑制剂受体的显性失活变体，或者所述分离的核酸能够提供所述受体的显性失活变体免疫抑制剂给宿主细胞。
8.根据实施例1-2中任一项所述的分离的核酸序列或修饰的细胞，其中所述CCR的细胞外结构域包含SEQ ID NO：45或47的氨基酸序列，所述CCD的跨膜结构域包含氨基酸SEQID NO：49或51的序列，和/或CCR的胞内结构域包含SEQ ID NO：53或55的氨基酸序列。
9.根据实施例1-8中任一项所述的分离的核酸序列或修饰的细胞，其中所述修饰的细胞包含嵌合抗原受体(CAR)或所述分离的核酸序列包含编码CAR的核酸序列。
10.实施例9的分离的核酸序列或修饰的细胞，其中所述CAR包含细胞外结构域，跨膜结构域和细胞内结构域，所述细胞外结构域结合抗原。
11.实施例10的分离的核酸序列或修饰的细胞，其中所述胞内结构域包含共刺激信号传导区，所述共刺激信号传导区包含选自CD27，CD28,4-1BB，OX40，CD30，CD40，PD-1，ICOS，淋巴细胞功能相关抗原-1(LFA-1)，CD2，CD7，LIGHT，NKG2C，B7-H3及其任何组合。
12.实施例9的分离的核酸序列或修饰的细胞，其中抗原选自表皮生长因子受体(EGFR)，表皮生长因子受体(EGFRvIII)的变体III，人表皮生长因子受体2(HER2)，间皮素(MSLN)，前列腺特异性膜抗原(PSMA)，癌胚抗原(CEA)，二糖神经节苷脂2(GD2)，白介素-13Ra2(IL13Rα2)，磷脂酰肌醇蛋白聚糖-3(GPC3)，碳酸酐酶IX(CAIX)，L1细胞粘附分子(L1-CAM)，癌抗原125(CA125)，分化簇133(CD133)，成纤维细胞活化蛋白(FAP)，癌/睾丸抗原1B(CTAG1B)，粘蛋白1(MUC1)，叶酸受体-α(FR-α)CD19，FZD10，TSHR，PRLR，Muc17，GUCY2C，CD207，CD3，CD5，B细胞成熟抗原(BCMA)或CD4。
13.一种CAR细胞群，其包含实施例1-12中任一项的分离的核酸序列。
14.实施例1-8中任一项的分离的核酸序列或修饰的细胞，其中所述修饰的细胞包含修饰的T细胞受体(TCR)或所述分离的核酸序列包含编码所述修饰的TCR的核酸序列。
15.根据实施例14所述的分离的核酸序列或修饰的细胞，其中所述TCR源自患者中自发发生的肿瘤特异性T细胞。
16.实施例14的分离的核酸序列或修饰的细胞，其中所述TCR结合肿瘤抗原。
17.实施例16的分离的核酸序列或修饰的细胞，其中所述肿瘤抗原包含CEA，gp100，MART-1，p53，MAGE-A3或NY-ESO-1。
18.实施例16的分离的核酸序列或修饰的细胞，其中所述TCR包含TCRγ和TCRδ链或TCRα和TCRβ链或其组合。
19.一种药物组合物，其包含实施例1-12中任一项的CAR细胞群。
20.根据实施例1-19中任一项所述的分离的核酸序列或修饰的细胞，其中所述修饰的细胞包含SEQ ID NO：56-67的任一氨基酸序列。
嵌合趋化因子受体(CCR)载体在293T上的表达
图6是已构建嵌合趋化因子受体载体列表。嵌合趋化因子受体就是胞外用PD1的胞外段，跨膜用PD1或别的因子受体的跨膜段，胞内用因子受体的胞内段，做成的嵌合受体。列表上构建的因子受体分别用到了IL7receptorα，GP130，CD27，IL2receptorγ，IL2/15receptorβ。所有的相关的序列在表2和表3-1至3-3。
图7-10显示各载体的PD1和CAR在293T细胞中都是呈双阳性表达的。实验方法：Day0在293T细胞感染对应慢病毒载体，Day1换液，Day3流式检测CAR和PD1的表达。CAR是人源化CD19CAR，PD1是嵌合受体的胞外段PD1。可以从图中看出，构建的10种CCR载体都能在293T细胞上表达。
嵌合趋化因子受体(CCR)载体在T细胞上的表达
图11-14显示嵌合趋化因子受体(CCR)载体在T细胞上的表达。实验方法：Day0抽取健康志愿者外周血，分选CD3+T细胞，按照1：1比例加入CD3/CD28Dynabeads。Day1感染T细胞，Day2换液，去掉慢病毒和beads，用新鲜的培养基重悬。
Day6流式检测CAR和PD1。CAR是人源化CD19CAR，PD1代表嵌合受体的表达情况。实验结果：进行了两次感染实验，一次是用“各受体跨膜”载体感染，一次是用“PD1跨膜‘载体感染。阳性对照19CAR只有CAR表达，无PD1/CAR双阳性细胞。阳性对照19CAR-2A-truncatePD1有明显的CAR/PD1双阳性细胞。在使用“各受体跨膜”的5种载体中，只有CD27组有PD1/CAR双阳性细胞。在使用“PD1跨膜”的5种载体中，CD27组有明显的PD1/CAR双阳性细胞，另外四种载体较“各受体跨膜”组有较好的PD1/CAR双阳性表达。因此，在用“各受体跨膜”和“PD1跨膜”的结构中，CD27嵌合受体表达是比较好的，其他受体在使用“PD1跨膜”结构时表达好一些。
CAR-CCR T细胞和Nalm6或Nalm6-PDL1细胞
图15显示CAR-CCR T细胞和Nalm6或Nalm6-PDL1细胞共培养的肿瘤细胞数量曲线。实验方法：Day6流式检测完表达后，用106个T细胞(CFSE标记)和106个Nalm6或Nalm6-PDL1肿瘤细胞(GFP标记)共培养，3天后(96h)，流式细胞计数检测残余的肿瘤细胞数。实验结果：这一次实验用的都是带“各受体跨膜”结构的CCR。图3左边代表的是和Nalm6共培养的肿瘤细胞数量曲线，右边是和Nalm6-PDL1共培养的肿瘤细胞数量曲线。各组起始的肿瘤细胞数一致(0d时)，第三天检测时除NT外都对肿瘤细胞有明显杀伤。和Nalm6共培养的杀伤效果显示CD27，IL2/15RB，IL2RG三种杀伤肿瘤细胞最多，tPD1，19CAR，GP130，IL7RA杀伤效果较弱。和Nalm6-PDL1共培养的杀伤效果显示CD27，IL2/15RB，tPD1，IL2RG杀伤肿瘤细胞最多，19CAR，GP130杀伤较少，IL7RA杀伤最少。从3天的杀伤结果来看，在用各受体自带跨膜结构的载体中，CD27，IL2/15RB，IL2RG这三种杀伤效果比较好。
图16显示CAR-CCR T细胞和Nalm6-PDL1细胞共培养7天的肿瘤细胞数量曲线。实验方法：Day6流式检测完表达后，用106个T细胞(CFSE标记)和106个Nalm6-PDL1肿瘤细胞(GFP标记)共培养，3天后(96h)流式计数检测肿瘤细胞数，并且各补充106个Nalm6-PDL1肿瘤细胞，2天后(48h，也就是共培养第5天)，4天后(也就是共培养第7天)，流式细胞计数检测残余的肿瘤细胞数。实验结果：第3天补充Nalm6-PDL1细胞后算作第二轮共培养。阴性对照NT不能杀伤，因此肿瘤细胞继续生长。其他CART继续杀伤肿瘤细胞，肿瘤细胞数目持续减少。CD27组在7d时基本将肿瘤杀光，GP130组对肿瘤细胞杀伤最少。此为第二轮杀伤，因此起始的肿瘤细胞数是不同的。从可比较的组别来看，tPD1组起始肿瘤细胞数比CD27组少，而7d时CD27组残留的肿瘤细胞数比tPD1少，这说明CD27组的杀伤能力强于tPD1。IL2RG和IL2/15RB组起始和7d的肿瘤细胞数都一致，说明杀伤能力一样。GP130组和IL7RA组起始肿瘤细胞数一样，7d时IL7RA残留肿瘤细胞数少于GP130组，说明IL7RA组杀伤强于GP130组。
CAR-CCR T细胞和Nalm6/Nalm6-PDL1细胞共培养
图17-22显示CAR-CCR T细胞和Nalm6/Nalm6-PDL1细胞共培养24h的CD137表达。实验方法：Day6流式检测完表达后，用106个T细胞和106个Nalm6或Nalm6-PDL1肿瘤细胞共培养，24h收细胞检测CAR+CD137。CD137代表CART激活。实验结果：如图所示，静息状态下(Alone)，细胞都没有CD137表达。于Nalm6或Nalm6-PDL1共培养后都有CD137表达。CD137表达代表了CART激活。因此次实验一方面证明了CAR-CCR T细胞能够激活，另一方面利用CD137表达的比例推算CAR阳性细胞的比例。结果如表1各CAR-CCR T激活后CD137比例：
表1细胞共培养的CD137表达
图23和24显示CAR-CCR T细胞和Nalm6/Nalm6-PDL1细胞共培养24/48h的细胞因子释放水平。实验方法：Day6流式检测完表达后，用106个T细胞和106个Nalm6或Nalm6-PDL1肿瘤细胞共培养，24h和48h后收取上清检测IL2，IL6，TNFα，IFNγ。实验结果：用上清的因子浓度和表1的激活细胞比例及数量推算出每1万个激活的CART细胞所释放的因子数，如图23和24。首先所有的CART细胞都有因子释放证明了其激活。所有跟Nalm6-PDL1共培养得到的因子数都比相同细胞和Nalm6共培养释放的因子少，说明了PDL1的抑制作用。所有48h的因子数目都比24h多，说明了因子的释放和累积。在和不带PDL1的Nalm6共培养的情况下，GP130组有最高的因子释放，在和带PDL1的肿瘤细胞共培养中，没有哪一组构建是有明显的细胞因子释放优势的。CD27组和tPD1组有类似的CD137激活比例。
表2：序列编号和其名称
表3-1序列编号-1
表3-2序列编号-2
表3-3序列编号-3
本说明书中引用的所有出版物，专利和专利申请均通过引用整体并入本文，如同每个单独的出版物，专利或专利申请被具体和单独地指示为通过引用并入。虽然已经根据各种实施例描述了前述内容，但是本领域技术人员将认识到，在不脱离其精神的情况下可以进行各种修改，替换，省略和改变。
序列表
<110> 上海斯丹赛生物技术有限公司
<120> 嵌合细胞因子受体
<150> US62/688,105
<151> 2018-06-21
<160> 67
<170> SIPOSequenceListing 1.0
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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
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Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu
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Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
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Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Val Arg
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Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Trp Gly Ser
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Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Phe Thr Ile Ser
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Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg
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Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
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Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
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Ser Ser
<210> 19
<211> 242
<212> PRT
<213> scFv Humanized CD19
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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
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Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
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Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 20
<211> 237
<212> PRT
<213> scFv FZD10
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Val Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Gly Gly Gly Gly Ser Gly Gly Gly
100 105 110
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Ala
115 120 125
Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser
130 135 140
Gly Phe Asn Ile Asn Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro
145 150 155 160
Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn
165 170 175
Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp
180 185 190
Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu
195 200 205
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala Arg Gly Ser Arg Phe
210 215 220
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
225 230 235
<210> 21
<211> 244
<212> PRT
<213> scFv TSHR
<400> 21
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln
1 5 10 15
Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Ile Gly Ser Asn
20 25 30
Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Asn Asn Lys Arg Pro Ser Ala Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln
65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Arg Leu
85 90 95
Gly Ile Ala Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
115 120 125
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gln Ser Leu Lys
130 135 140
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Asp Asn Trp Ile Gly
145 150 155 160
Trp Val Arg Gln Lys Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile
165 170 175
Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln
180 185 190
Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr Leu Gln Trp
195 200 205
Ser Ser Leu Lys Ala Ser Asp Thr Ala Ile Tyr Tyr Cys Val Gly Leu
210 215 220
Asp Trp Asn Tyr Asn Pro Leu Arg Tyr Trp Gly Pro Gly Thr Leu Val
225 230 235 240
Thr Val Ser Ser
<210> 22
<211> 252
<212> PRT
<213> scFv PRLR
<400> 22
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Gly Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Gly
85 90 95
Glu Leu Pro Pro Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
130 135 140
Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr
145 150 155 160
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
165 170 175
Ala Thr Val Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Ser Ala Met Tyr Tyr Cys
210 215 220
Ala Arg His Arg Gly Asn Tyr Tyr Ala Thr Tyr Tyr Tyr Ala Met Asp
225 230 235 240
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
245 250
<210> 23
<211> 243
<212> PRT
<213> scFv Muc 17
<400> 23
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His His Glu Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln
115 120 125
Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys
130 135 140
Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Trp Met Asn Trp Val Lys
145 150 155 160
Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr
165 170 175
Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu
180 185 190
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu
195 200 205
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Pro Tyr Tyr
210 215 220
Gly Thr Asn Pro Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
<210> 24
<211> 241
<212> PRT
<213> scFv GUCY2C
<400> 24
Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Thr Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln
115 120 125
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
130 135 140
Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Arg
165 170 175
Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser
180 185 190
Val Asp Thr Ser Lys Asn Gln Phe Ala Leu Lys Leu Ser Ser Val Thr
195 200 205
Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Arg Gly Tyr Thr
210 215 220
Tyr Gly Asn Phe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser
225 230 235 240
Ser
<210> 25
<211> 246
<212> PRT
<213> scFv CD207
<400> 25
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser
85 90 95
Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gln Val Gln Leu Arg Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
130 135 140
Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
145 150 155 160
Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp
165 170 175
Ile Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser Phe Tyr Asn Glu Asn
180 185 190
Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala
195 200 205
Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
210 215 220
Cys Ala Thr Tyr Tyr Asn Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr
225 230 235 240
Leu Val Thr Val Ser Ala
245
<210> 26
<211> 199
<212> PRT
<213> Prolactin ligand
<400> 26
Leu Pro Ile Cys Pro Gly Gly Ala Ala Arg Cys Gln Val Thr Leu Arg
1 5 10 15
Asp Leu Phe Asp Arg Ala Val Val Leu Ser His Tyr Ile His Asn Leu
20 25 30
Ser Ser Glu Met Phe Ser Glu Phe Asp Lys Arg Tyr Thr His Gly Arg
35 40 45
Gly Phe Ile Thr Lys Ala Ile Asn Ser Cys His Thr Ser Ser Leu Ala
50 55 60
Thr Pro Glu Asp Lys Glu Gln Ala Gln Gln Met Asn Gln Lys Asp Phe
65 70 75 80
Leu Ser Leu Ile Val Ser Ile Leu Arg Ser Trp Asn Glu Pro Leu Tyr
85 90 95
His Leu Val Thr Glu Val Arg Gly Met Gln Glu Ala Pro Glu Ala Ile
100 105 110
Leu Ser Lys Ala Val Glu Ile Glu Glu Gln Thr Lys Arg Leu Leu Glu
115 120 125
Gly Met Glu Leu Ile Val Ser Gln Val His Pro Glu Thr Lys Glu Asn
130 135 140
Glu Ile Tyr Pro Val Trp Ser Gly Leu Pro Ser Leu Gln Met Ala Asp
145 150 155 160
Glu Glu Ser Arg Leu Ser Ala Tyr Tyr Asn Leu Leu His Cys Leu Arg
165 170 175
Arg Asp Ser His Lys Ile Asp Asn Tyr Leu Lys Leu Leu Lys Cys Arg
180 185 190
Ile Ile His Asn Asn Asn Cys
195
<210> 27
<211> 240
<212> PRT
<213> scFv CD3
<400> 27
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln
85 90 95
Ser Phe Ile Leu Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
130 135 140
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
145 150 155 160
Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
165 170 175
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
180 185 190
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln
210 215 220
Ser Phe Ile Leu Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
225 230 235 240
<210> 28
<211> 281
<212> PRT
<213> scFv CD4 1
<400> 28
Met Asn Val Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Gly Gly Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser
20 25 30
Ala Ser Leu Glu Glu Ile Val Thr Ile Thr Cys Lys Ala Ser Gln Ala
35 40 45
Ile Asp Ala Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro
50 55 60
Gln Leu Leu Ile Tyr Asp Ala Thr Ser Leu Ala Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Arg Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser
85 90 95
Arg Pro Gln Val Asp Asp Ser Gly Ile Tyr Tyr Cys Leu Gln Ser Tyr
100 105 110
Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met
130 135 140
Ala Val Leu Val Leu Leu Leu Cys Leu Leu Ile Phe Pro Ser Cys Val
145 150 155 160
Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro
165 170 175
Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Thr
180 185 190
Ser Asn Ser Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
195 200 205
Trp Met Gly Val Ile Trp Ser Asn Gly Asp Ala Asp Tyr Asn Ser Ala
210 215 220
Ile Lys Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val
225 230 235 240
Phe Leu Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Met Tyr Phe
245 250 255
Cys Ala Ser Pro Tyr Tyr Gly Tyr Tyr Phe Pro Phe Asp Tyr Trp Gly
260 265 270
Gln Gly Val Met Val Thr Val Ser Ser
275 280
<210> 29
<211> 285
<212> PRT
<213> scFv CD4 2
<400> 29
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val
20 25 30
Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val
35 40 45
Ser Ile Ser Ser His Asp Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly
50 55 60
Gln Gln Pro Lys Leu Leu Ile Tyr Asp Ala Phe Asn Leu Ala Ser Gly
65 70 75 80
Ile Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
85 90 95
Thr Ile Asp Pro Val Gln Ala Asp Asp Ile Ala Thr Tyr Tyr Cys Gln
100 105 110
Gln Ser Lys Asp Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu
115 120 125
Leu Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Met Asp Ile Arg Leu Ser Leu Ala Phe Leu Val Leu Phe Ile
145 150 155 160
Lys Gly Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
165 170 175
Val Gln Pro Gly Arg Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe
180 185 190
Thr Phe Ser Asn Tyr Gly Met Ala Trp Val Arg Gln Ala Pro Thr Lys
195 200 205
Gly Leu Glu Trp Val Ala Thr Ile Ser Tyr Asp Gly Ser Ile Thr Tyr
210 215 220
Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ala
225 230 235 240
Lys Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr
245 250 255
Ala Thr Tyr Tyr Cys Thr Arg Glu Glu Gln Tyr Ser Ser Trp Tyr Phe
260 265 270
Asp Phe Trp Gly Pro Gly Ile Met Val Thr Val Ser Ser
275 280 285
<210> 30
<211> 242
<212> PRT
<213> scFv CD5
<400> 30
Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr 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 Ile Ala Thr Tyr Phe Cys His Gln Tyr Asn Ser Tyr Asn Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu
115 120 125
Ser Gly Pro Val Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys
130 135 140
Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp
145 150 155 160
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala His Ile Trp
165 170 175
Trp Asp Asp Asp Val Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr
180 185 190
Ile Thr Lys Asp Ala Ser Lys Asp Gln Val Ser Leu Lys Leu Ser Ser
195 200 205
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val Arg Arg Arg Ala
210 215 220
Thr Gly Thr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser
<210> 31
<211> 244
<212> PRT
<213> ScFv MUC1-5e5
<400> 31
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ile Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ser
130 135 140
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His
145 150 155 160
Ala Ile His Trp Val Lys Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile
165 170 175
Gly His Phe Ser Pro Gly Asn Thr Asp Ile Lys Tyr Asn Asp Lys Phe
180 185 190
Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr
195 200 205
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
210 215 220
Lys Thr Ser Thr Phe Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
225 230 235 240
Thr Val Ser Ser
<210> 32
<211> 246
<212> PRT
<213> ScFv MUC1-Panko
<400> 32
Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
1 5 10 15
Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys Pro Gly Leu Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
85 90 95
Leu Glu Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
130 135 140
Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn
145 150 155 160
Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp
165 170 175
Val Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala
180 185 190
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
195 200 205
Ser Val Ser Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Gly Ile
210 215 220
Tyr Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
225 230 235 240
Thr Leu Thr Val Ser Ser
245
<210> 33
<211> 272
<212> PRT
<213> CD19 antigen
<400> 33
Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala Val
1 5 10 15
Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu Thr
20 25 30
Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu Gly
35 40 45
Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu Phe
50 55 60
Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln Pro
65 70 75 80
Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val
85 90 95
Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly
100 105 110
Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro
115 120 125
Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg
130 135 140
Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp Ser
145 150 155 160
Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser Thr
165 170 175
Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly Pro
180 185 190
Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu Ser
195 200 205
Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu
210 215 220
Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr
225 230 235 240
Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr
245 250 255
Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp Lys
260 265 270
<210> 34
<211> 205
<212> PRT
<213> FZD10 antigen
<400> 34
Ile Ser Ser Met Asp Met Glu Arg Pro Gly Asp Gly Lys Cys Gln Pro
1 5 10 15
Ile Glu Ile Pro Met Cys Lys Asp Ile Gly Tyr Asn Met Thr Arg Met
20 25 30
Pro Asn Leu Met Gly His Glu Asn Gln Arg Glu Ala Ala Ile Gln Leu
35 40 45
His Glu Phe Ala Pro Leu Val Glu Tyr Gly Cys His Gly His Leu Arg
50 55 60
Phe Phe Leu Cys Ser Leu Tyr Ala Pro Met Cys Thr Glu Gln Val Ser
65 70 75 80
Thr Pro Ile Pro Ala Cys Arg Val Met Cys Glu Gln Ala Arg Leu Lys
85 90 95
Cys Ser Pro Ile Met Glu Gln Phe Asn Phe Lys Trp Pro Asp Ser Leu
100 105 110
Asp Cys Arg Lys Leu Pro Asn Lys Asn Asp Pro Asn Tyr Leu Cys Met
115 120 125
Glu Ala Pro Asn Asn Gly Ser Asp Glu Pro Thr Arg Gly Ser Gly Leu
130 135 140
Phe Pro Pro Leu Phe Arg Pro Gln Arg Pro His Ser Ala Gln Glu His
145 150 155 160
Pro Leu Lys Asp Gly Gly Pro Gly Arg Gly Gly Cys Asp Asn Pro Gly
165 170 175
Lys Phe His His Val Glu Lys Ser Ala Ser Cys Ala Pro Leu Cys Thr
180 185 190
Pro Gly Val Asp Val Tyr Trp Ser Arg Glu Asp Lys Arg
195 200 205
<210> 35
<211> 393
<212> PRT
<213> TSHR antigen
<400> 35
Gly Met Gly Cys Ser Ser Pro Pro Cys Glu Cys His Gln Glu Glu Asp
1 5 10 15
Phe Arg Val Thr Cys Lys Asp Ile Gln Arg Ile Pro Ser Leu Pro Pro
20 25 30
Ser Thr Gln Thr Leu Lys Leu Ile Glu Thr His Leu Arg Thr Ile Pro
35 40 45
Ser His Ala Phe Ser Asn Leu Pro Asn Ile Ser Arg Ile Tyr Val Ser
50 55 60
Ile Asp Val Thr Leu Gln Gln Leu Glu Ser His Ser Phe Tyr Asn Leu
65 70 75 80
Ser Lys Val Thr His Ile Glu Ile Arg Asn Thr Arg Asn Leu Thr Tyr
85 90 95
Ile Asp Pro Asp Ala Leu Lys Glu Leu Pro Leu Leu Lys Phe Leu Gly
100 105 110
Ile Phe Asn Thr Gly Leu Lys Met Phe Pro Asp Leu Thr Lys Val Tyr
115 120 125
Ser Thr Asp Ile Phe Phe Ile Leu Glu Ile Thr Asp Asn Pro Tyr Met
130 135 140
Thr Ser Ile Pro Val Asn Ala Phe Gln Gly Leu Cys Asn Glu Thr Leu
145 150 155 160
Thr Leu Lys Leu Tyr Asn Asn Gly Phe Thr Ser Val Gln Gly Tyr Ala
165 170 175
Phe Asn Gly Thr Lys Leu Asp Ala Val Tyr Leu Asn Lys Asn Lys Tyr
180 185 190
Leu Thr Val Ile Asp Lys Asp Ala Phe Gly Gly Val Tyr Ser Gly Pro
195 200 205
Ser Leu Leu Asp Val Ser Gln Thr Ser Val Thr Ala Leu Pro Ser Lys
210 215 220
Gly Leu Glu His Leu Lys Glu Leu Ile Ala Arg Asn Thr Trp Thr Leu
225 230 235 240
Lys Lys Leu Pro Leu Ser Leu Ser Phe Leu His Leu Thr Arg Ala Asp
245 250 255
Leu Ser Tyr Pro Ser His Cys Cys Ala Phe Lys Asn Gln Lys Lys Ile
260 265 270
Arg Gly Ile Leu Glu Ser Leu Met Cys Asn Glu Ser Ser Met Gln Ser
275 280 285
Leu Arg Gln Arg Lys Ser Val Asn Ala Leu Asn Ser Pro Leu His Gln
290 295 300
Glu Tyr Glu Glu Asn Leu Gly Asp Ser Ile Val Gly Tyr Lys Glu Lys
305 310 315 320
Ser Lys Phe Gln Asp Thr His Asn Asn Ala His Tyr Tyr Val Phe Phe
325 330 335
Glu Glu Gln Glu Asp Glu Ile Ile Gly Phe Gly Gln Glu Leu Lys Asn
340 345 350
Pro Gln Glu Glu Thr Leu Gln Ala Phe Asp Ser His Tyr Asp Tyr Thr
355 360 365
Ile Cys Gly Asp Ser Glu Asp Met Val Cys Thr Pro Lys Ser Asp Glu
370 375 380
Phe Asn Pro Cys Glu Asp Ile Met Gly
385 390
<210> 36
<211> 210
<212> PRT
<213> PRLR antigen
<400> 36
Gln Leu Pro Pro Gly Lys Pro Glu Ile Phe Lys Cys Arg Ser Pro Asn
1 5 10 15
Lys Glu Thr Phe Thr Cys Trp Trp Arg Pro Gly Thr Asp Gly Gly Leu
20 25 30
Pro Thr Asn Tyr Ser Leu Thr Tyr His Arg Glu Gly Glu Thr Leu Met
35 40 45
His Glu Cys Pro Asp Tyr Ile Thr Gly Gly Pro Asn Ser Cys His Phe
50 55 60
Gly Lys Gln Tyr Thr Ser Met Trp Arg Thr Tyr Ile Met Met Val Asn
65 70 75 80
Ala Thr Asn Gln Met Gly Ser Ser Phe Ser Asp Glu Leu Tyr Val Asp
85 90 95
Val Thr Tyr Ile Val Gln Pro Asp Pro Pro Leu Glu Leu Ala Val Glu
100 105 110
Val Lys Gln Pro Glu Asp Arg Lys Pro Tyr Leu Trp Ile Lys Trp Ser
115 120 125
Pro Pro Thr Leu Ile Asp Leu Lys Thr Gly Trp Phe Thr Leu Leu Tyr
130 135 140
Glu Ile Arg Leu Lys Pro Glu Lys Ala Ala Glu Trp Glu Ile His Phe
145 150 155 160
Ala Gly Gln Gln Thr Glu Phe Lys Ile Leu Ser Leu His Pro Gly Gln
165 170 175
Lys Tyr Leu Val Gln Val Arg Cys Lys Pro Asp His Gly Tyr Trp Ser
180 185 190
Ala Trp Ser Pro Ala Thr Phe Ile Gln Ile Pro Ser Asp Phe Thr Met
195 200 205
Asn Asp
210
<210> 37
<211> 4368
<212> PRT
<213> Muc 17 antigen
<400> 37
Glu Gln Asp Leu Ser Val Asn Arg Ala Val Trp Asp Gly Gly Gly Cys
1 5 10 15
Ile Ser Gln Gly Asp Val Leu Asn Arg Gln Cys Gln Gln Leu Ser Gln
20 25 30
His Val Arg Thr Gly Ser Ala Ala Asn Thr Ala Thr Gly Thr Thr Ser
35 40 45
Thr Asn Val Val Glu Pro Arg Met Tyr Leu Ser Cys Ser Thr Asn Pro
50 55 60
Glu Met Thr Ser Ile Glu Ser Ser Val Thr Ser Asp Thr Pro Gly Val
65 70 75 80
Ser Ser Thr Arg Met Thr Pro Thr Glu Ser Arg Thr Thr Ser Glu Ser
85 90 95
Thr Ser Asp Ser Thr Thr Leu Phe Pro Ser Ser Thr Glu Asp Thr Ser
100 105 110
Ser Pro Thr Thr Pro Glu Gly Thr Asp Val Pro Met Ser Thr Pro Ser
115 120 125
Glu Glu Ser Ile Ser Ser Thr Met Ala Phe Val Ser Thr Ala Pro Leu
130 135 140
Pro Ser Phe Glu Ala Tyr Thr Ser Leu Thr Tyr Lys Val Asp Met Ser
145 150 155 160
Thr Pro Leu Thr Thr Ser Thr Gln Ala Ser Ser Ser Pro Thr Thr Pro
165 170 175
Glu Ser Thr Thr Ile Pro Lys Ser Thr Asn Ser Glu Gly Ser Thr Pro
180 185 190
Leu Thr Ser Met Pro Ala Ser Thr Met Lys Val Ala Ser Ser Glu Ala
195 200 205
Ile Thr Leu Leu Thr Thr Pro Val Glu Ile Ser Thr Pro Val Thr Ile
210 215 220
Ser Ala Gln Ala Ser Ser Ser Pro Thr Thr Ala Glu Gly Pro Ser Leu
225 230 235 240
Ser Asn Ser Ala Pro Ser Gly Gly Ser Thr Pro Leu Thr Arg Met Pro
245 250 255
Leu Ser Val Met Leu Val Val Ser Ser Glu Ala Ser Thr Leu Ser Thr
260 265 270
Thr Pro Ala Ala Thr Asn Ile Pro Val Ile Thr Ser Thr Glu Ala Ser
275 280 285
Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Ile Pro Thr Ser Thr Tyr
290 295 300
Thr Glu Gly Ser Thr Pro Leu Thr Ser Thr Pro Ala Ser Thr Met Pro
305 310 315 320
Val Ala Thr Ser Glu Met Ser Thr Leu Ser Ile Thr Pro Val Asp Thr
325 330 335
Ser Thr Leu Val Thr Thr Ser Thr Glu Pro Ser Ser Leu Pro Thr Thr
340 345 350
Ala Glu Ala Thr Ser Met Leu Thr Ser Thr Leu Ser Glu Gly Ser Thr
355 360 365
Pro Leu Thr Asn Met Pro Val Ser Thr Ile Leu Val Ala Ser Ser Glu
370 375 380
Ala Ser Thr Thr Ser Thr Ile Pro Val Asp Ser Lys Thr Phe Val Thr
385 390 395 400
Thr Ala Ser Glu Ala Ser Ser Ser Pro Thr Thr Ala Glu Asp Thr Ser
405 410 415
Ile Ala Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Ser Met
420 425 430
Pro Val Ser Thr Thr Pro Val Ala Ser Ser Glu Ala Ser Asn Leu Ser
435 440 445
Thr Thr Pro Val Asp Ser Lys Thr Gln Val Thr Thr Ser Thr Glu Ala
450 455 460
Ser Ser Ser Pro Pro Thr Ala Glu Val Asn Ser Met Pro Thr Ser Thr
465 470 475 480
Pro Ser Glu Gly Ser Thr Pro Leu Thr Ser Met Ser Val Ser Thr Met
485 490 495
Pro Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp
500 505 510
Thr Ser Thr Pro Val Thr Thr Ser Ser Glu Ala Ser Ser Ser Ser Thr
515 520 525
Thr Pro Glu Gly Thr Ser Ile Pro Thr Ser Thr Pro Ser Glu Gly Ser
530 535 540
Thr Pro Leu Thr Asn Met Pro Val Ser Thr Arg Leu Val Val Ser Ser
545 550 555 560
Glu Ala Ser Thr Thr Ser Thr Thr Pro Ala Asp Ser Asn Thr Phe Val
565 570 575
Thr Thr Ser Ser Glu Ala Ser Ser Ser Ser Thr Thr Ala Glu Gly Thr
580 585 590
Ser Met Pro Thr Ser Thr Tyr Ser Glu Arg Gly Thr Thr Ile Thr Ser
595 600 605
Met Ser Val Ser Thr Thr Leu Val Ala Ser Ser Glu Ala Ser Thr Leu
610 615 620
Ser Thr Thr Pro Val Asp Ser Asn Thr Pro Val Thr Thr Ser Thr Glu
625 630 635 640
Ala Thr Ser Ser Ser Thr Thr Ala Glu Gly Thr Ser Met Pro Thr Ser
645 650 655
Thr Tyr Thr Glu Gly Ser Thr Pro Leu Thr Ser Met Pro Val Asn Thr
660 665 670
Thr Leu Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
675 680 685
Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro
690 695 700
Thr Thr Ala Asp Gly Ala Ser Met Pro Thr Ser Thr Pro Ser Glu Gly
705 710 715 720
Ser Thr Pro Leu Thr Ser Met Pro Val Ser Lys Thr Leu Leu Thr Ser
725 730 735
Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Leu Asp Thr Ser Thr His
740 745 750
Ile Thr Thr Ser Thr Glu Ala Ser Cys Ser Pro Thr Thr Thr Glu Gly
755 760 765
Thr Ser Met Pro Ile Ser Thr Pro Ser Glu Gly Ser Pro Leu Leu Thr
770 775 780
Ser Ile Pro Val Ser Ile Thr Pro Val Thr Ser Pro Glu Ala Ser Thr
785 790 795 800
Leu Ser Thr Thr Pro Val Asp Ser Asn Ser Pro Val Thr Thr Ser Thr
805 810 815
Glu Val Ser Ser Ser Pro Thr Pro Ala Glu Gly Thr Ser Met Pro Thr
820 825 830
Ser Thr Tyr Ser Glu Gly Arg Thr Pro Leu Thr Ser Met Pro Val Ser
835 840 845
Thr Thr Leu Val Ala Thr Ser Ala Ile Ser Thr Leu Ser Thr Thr Pro
850 855 860
Val Asp Thr Ser Thr Pro Val Thr Asn Ser Thr Glu Ala Arg Ser Ser
865 870 875 880
Pro Thr Thr Ser Glu Gly Thr Ser Met Pro Thr Ser Thr Pro Gly Glu
885 890 895
Gly Ser Thr Pro Leu Thr Ser Met Pro Asp Ser Thr Thr Pro Val Val
900 905 910
Ser Ser Glu Ala Arg Thr Leu Ser Ala Thr Pro Val Asp Thr Ser Thr
915 920 925
Pro Val Thr Thr Ser Thr Glu Ala Thr Ser Ser Pro Thr Thr Ala Glu
930 935 940
Gly Thr Ser Ile Pro Thr Ser Thr Pro Ser Glu Gly Thr Thr Pro Leu
945 950 955 960
Thr Ser Thr Pro Val Ser His Thr Leu Val Ala Asn Ser Glu Ala Ser
965 970 975
Thr Leu Ser Thr Thr Pro Val Asp Ser Asn Thr Pro Leu Thr Thr Ser
980 985 990
Thr Glu Ala Ser Ser Pro Pro Pro Thr Ala Glu Gly Thr Ser Met Pro
995 1000 1005
Thr Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Arg Met Pro Val
1010 1015 1020
Ser Thr Thr Met Val Ala Ser Ser Glu Thr Ser Thr Leu Ser Thr Thr
1025 1030 1035 1040
Pro Ala Asp Thr Ser Thr Pro Val Thr Thr Tyr Ser Gln Ala Ser Ser
1045 1050 1055
Ser Ser Thr Thr Ala Asp Gly Thr Ser Met Pro Thr Ser Thr Tyr Ser
1060 1065 1070
Glu Gly Ser Thr Pro Leu Thr Ser Val Pro Val Ser Thr Arg Leu Val
1075 1080 1085
Val Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser
1090 1095 1100
Ile Pro Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala
1105 1110 1115 1120
Glu Gly Thr Ser Ile Pro Thr Ser Pro Pro Ser Glu Gly Thr Thr Pro
1125 1130 1135
Leu Ala Ser Met Pro Val Ser Thr Thr Leu Val Val Ser Ser Glu Ala
1140 1145 1150
Asn Thr Leu Ser Thr Thr Pro Val Asp Ser Lys Thr Gln Val Ala Thr
1155 1160 1165
Ser Thr Glu Ala Ser Ser Pro Pro Pro Thr Ala Glu Val Thr Ser Met
1170 1175 1180
Pro Thr Ser Thr Pro Gly Glu Arg Ser Thr Pro Leu Thr Ser Met Pro
1185 1190 1195 1200
Val Arg His Thr Pro Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr
1205 1210 1215
Ser Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser Ala Glu Thr Ser
1220 1225 1230
Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Leu Pro Thr Ser Thr Thr
1235 1240 1245
Ser Glu Gly Ser Thr Leu Leu Thr Ser Ile Pro Val Ser Thr Thr Leu
1250 1255 1260
Val Thr Ser Pro Glu Ala Ser Thr Leu Leu Thr Thr Pro Val Asp Thr
1265 1270 1275 1280
Lys Gly Pro Val Val Thr Ser Asn Glu Val Ser Ser Ser Pro Thr Pro
1285 1290 1295
Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Tyr Ser Glu Gly Arg Thr
1300 1305 1310
Pro Leu Thr Ser Ile Pro Val Asn Thr Thr Leu Val Ala Ser Ser Ala
1315 1320 1325
Ile Ser Ile Leu Ser Thr Thr Pro Val Asp Asn Ser Thr Pro Val Thr
1330 1335 1340
Thr Ser Thr Glu Ala Cys Ser Ser Pro Thr Thr Ser Glu Gly Thr Ser
1345 1350 1355 1360
Met Pro Asn Ser Asn Pro Ser Glu Gly Thr Thr Pro Leu Thr Ser Ile
1365 1370 1375
Pro Val Ser Thr Thr Pro Val Val Ser Ser Glu Ala Ser Thr Leu Ser
1380 1385 1390
Ala Thr Pro Val Asp Thr Ser Thr Pro Gly Thr Thr Ser Ala Glu Ala
1395 1400 1405
Thr Ser Ser Pro Thr Thr Ala Glu Gly Ile Ser Ile Pro Thr Ser Thr
1410 1415 1420
Pro Ser Glu Gly Lys Thr Pro Leu Lys Ser Ile Pro Val Ser Asn Thr
1425 1430 1435 1440
Pro Val Ala Asn Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp
1445 1450 1455
Ser Asn Ser Pro Val Val Thr Ser Thr Ala Val Ser Ser Ser Pro Thr
1460 1465 1470
Pro Ala Glu Gly Thr Ser Ile Ala Ile Ser Thr Pro Ser Glu Gly Ser
1475 1480 1485
Thr Ala Leu Thr Ser Ile Pro Val Ser Thr Thr Thr Val Ala Ser Ser
1490 1495 1500
Glu Ile Asn Ser Leu Ser Thr Thr Pro Ala Val Thr Ser Thr Pro Val
1505 1510 1515 1520
Thr Thr Tyr Ser Gln Ala Ser Ser Ser Pro Thr Thr Ala Asp Gly Thr
1525 1530 1535
Ser Met Gln Thr Ser Thr Tyr Ser Glu Gly Ser Thr Pro Leu Thr Ser
1540 1545 1550
Leu Pro Val Ser Thr Met Leu Val Val Ser Ser Glu Ala Asn Thr Leu
1555 1560 1565
Ser Thr Thr Pro Ile Asp Ser Lys Thr Gln Val Thr Ala Ser Thr Glu
1570 1575 1580
Ala Ser Ser Ser Thr Thr Ala Glu Gly Ser Ser Met Thr Ile Ser Thr
1585 1590 1595 1600
Pro Ser Glu Gly Ser Pro Leu Leu Thr Ser Ile Pro Val Ser Thr Thr
1605 1610 1615
Pro Val Ala Ser Pro Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp
1620 1625 1630
Ser Asn Ser Pro Val Ile Thr Ser Thr Glu Val Ser Ser Ser Pro Thr
1635 1640 1645
Pro Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Tyr Thr Glu Gly Arg
1650 1655 1660
Thr Pro Leu Thr Ser Ile Thr Val Arg Thr Thr Pro Val Ala Ser Ser
1665 1670 1675 1680
Ala Ile Ser Thr Leu Ser Thr Thr Pro Val Asp Asn Ser Thr Pro Val
1685 1690 1695
Thr Thr Ser Thr Glu Ala Arg Ser Ser Pro Thr Thr Ser Glu Gly Thr
1700 1705 1710
Ser Met Pro Asn Ser Thr Pro Ser Glu Gly Thr Thr Pro Leu Thr Ser
1715 1720 1725
Ile Pro Val Ser Thr Thr Pro Val Leu Ser Ser Glu Ala Ser Thr Leu
1730 1735 1740
Ser Ala Thr Pro Ile Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu
1745 1750 1755 1760
Ala Thr Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Ile Pro Thr Ser
1765 1770 1775
Thr Leu Ser Glu Gly Met Thr Pro Leu Thr Ser Thr Pro Val Ser His
1780 1785 1790
Thr Leu Val Ala Asn Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
1795 1800 1805
Asp Ser Asn Ser Pro Val Val Thr Ser Thr Ala Val Ser Ser Ser Pro
1810 1815 1820
Thr Pro Ala Glu Gly Thr Ser Ile Ala Thr Ser Thr Pro Ser Glu Gly
1825 1830 1835 1840
Ser Thr Ala Leu Thr Ser Ile Pro Val Ser Thr Thr Thr Val Ala Ser
1845 1850 1855
Ser Glu Thr Asn Thr Leu Ser Thr Thr Pro Ala Val Thr Ser Thr Pro
1860 1865 1870
Val Thr Thr Tyr Ala Gln Val Ser Ser Ser Pro Thr Thr Ala Asp Gly
1875 1880 1885
Ser Ser Met Pro Thr Ser Thr Pro Arg Glu Gly Arg Pro Pro Leu Thr
1890 1895 1900
Ser Ile Pro Val Ser Thr Thr Thr Val Ala Ser Ser Glu Ile Asn Thr
1905 1910 1915 1920
Leu Ser Thr Thr Leu Ala Asp Thr Arg Thr Pro Val Thr Thr Tyr Ser
1925 1930 1935
Gln Ala Ser Ser Ser Pro Thr Thr Ala Asp Gly Thr Ser Met Pro Thr
1940 1945 1950
Pro Ala Tyr Ser Glu Gly Ser Thr Pro Leu Thr Ser Met Pro Leu Ser
1955 1960 1965
Thr Thr Leu Val Val Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro
1970 1975 1980
Val Asp Thr Ser Thr Pro Ala Thr Thr Ser Thr Glu Gly Ser Ser Ser
1985 1990 1995 2000
Pro Thr Thr Ala Gly Gly Thr Ser Ile Gln Thr Ser Thr Pro Ser Glu
2005 2010 2015
Arg Thr Thr Pro Leu Ala Gly Met Pro Val Ser Thr Thr Leu Val Val
2020 2025 2030
Ser Ser Glu Gly Asn Thr Leu Ser Thr Thr Pro Val Asp Ser Lys Thr
2035 2040 2045
Gln Val Thr Asn Ser Thr Glu Ala Ser Ser Ser Ala Thr Ala Glu Gly
2050 2055 2060
Ser Ser Met Thr Ile Ser Ala Pro Ser Glu Gly Ser Pro Leu Leu Thr
2065 2070 2075 2080
Ser Ile Pro Leu Ser Thr Thr Pro Val Ala Ser Pro Glu Ala Ser Thr
2085 2090 2095
Leu Ser Thr Thr Pro Val Asp Ser Asn Ser Pro Val Ile Thr Ser Thr
2100 2105 2110
Glu Val Ser Ser Ser Pro Ile Pro Thr Glu Gly Thr Ser Met Gln Thr
2115 2120 2125
Ser Thr Tyr Ser Asp Arg Arg Thr Pro Leu Thr Ser Met Pro Val Ser
2130 2135 2140
Thr Thr Val Val Ala Ser Ser Ala Ile Ser Thr Leu Ser Thr Thr Pro
2145 2150 2155 2160
Val Asp Thr Ser Thr Pro Val Thr Asn Ser Thr Glu Ala Arg Ser Ser
2165 2170 2175
Pro Thr Thr Ser Glu Gly Thr Ser Met Pro Thr Ser Thr Pro Ser Glu
2180 2185 2190
Gly Ser Thr Pro Phe Thr Ser Met Pro Val Ser Thr Met Pro Val Val
2195 2200 2205
Thr Ser Glu Ala Ser Thr Leu Ser Ala Thr Pro Val Asp Thr Ser Thr
2210 2215 2220
Pro Val Thr Thr Ser Thr Glu Ala Thr Ser Ser Pro Thr Thr Ala Glu
2225 2230 2235 2240
Gly Thr Ser Ile Pro Thr Ser Thr Leu Ser Glu Gly Thr Thr Pro Leu
2245 2250 2255
Thr Ser Ile Pro Val Ser His Thr Leu Val Ala Asn Ser Glu Val Ser
2260 2265 2270
Thr Leu Ser Thr Thr Pro Val Asp Ser Asn Thr Pro Phe Thr Thr Ser
2275 2280 2285
Thr Glu Ala Ser Ser Pro Pro Pro Thr Ala Glu Gly Thr Ser Met Pro
2290 2295 2300
Thr Ser Thr Ser Ser Glu Gly Asn Thr Pro Leu Thr Arg Met Pro Val
2305 2310 2315 2320
Ser Thr Thr Met Val Ala Ser Phe Glu Thr Ser Thr Leu Ser Thr Thr
2325 2330 2335
Pro Ala Asp Thr Ser Thr Pro Val Thr Thr Tyr Ser Gln Ala Gly Ser
2340 2345 2350
Ser Pro Thr Thr Ala Asp Asp Thr Ser Met Pro Thr Ser Thr Tyr Ser
2355 2360 2365
Glu Gly Ser Thr Pro Leu Thr Ser Val Pro Val Ser Thr Met Pro Val
2370 2375 2380
Val Ser Ser Glu Ala Ser Thr His Ser Thr Thr Pro Val Asp Thr Ser
2385 2390 2395 2400
Thr Pro Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala
2405 2410 2415
Glu Gly Thr Ser Ile Pro Thr Ser Pro Pro Ser Glu Gly Thr Thr Pro
2420 2425 2430
Leu Ala Ser Met Pro Val Ser Thr Thr Pro Val Val Ser Ser Glu Ala
2435 2440 2445
Gly Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Met Thr Thr
2450 2455 2460
Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala Glu Asp Ile Val Val
2465 2470 2475 2480
Pro Ile Ser Thr Ala Ser Glu Gly Ser Thr Leu Leu Thr Ser Ile Pro
2485 2490 2495
Val Ser Thr Thr Pro Val Ala Ser Pro Glu Ala Ser Thr Leu Ser Thr
2500 2505 2510
Thr Pro Val Asp Ser Asn Ser Pro Val Val Thr Ser Thr Glu Ile Ser
2515 2520 2525
Ser Ser Ala Thr Ser Ala Glu Gly Thr Ser Met Pro Thr Ser Thr Tyr
2530 2535 2540
Ser Glu Gly Ser Thr Pro Leu Arg Ser Met Pro Val Ser Thr Lys Pro
2545 2550 2555 2560
Leu Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr
2565 2570 2575
Ser Ile Pro Val Thr Thr Ser Thr Glu Thr Ser Ser Ser Pro Thr Thr
2580 2585 2590
Ala Lys Asp Thr Ser Met Pro Ile Ser Thr Pro Ser Glu Val Ser Thr
2595 2600 2605
Ser Leu Thr Ser Ile Leu Val Ser Thr Met Pro Val Ala Ser Ser Glu
2610 2615 2620
Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Arg Thr Leu Val Thr
2625 2630 2635 2640
Thr Ser Thr Gly Thr Ser Ser Ser Pro Thr Thr Ala Glu Gly Ser Ser
2645 2650 2655
Met Pro Thr Ser Thr Pro Gly Glu Arg Ser Thr Pro Leu Thr Asn Ile
2660 2665 2670
Leu Val Ser Thr Thr Leu Leu Ala Asn Ser Glu Ala Ser Thr Leu Ser
2675 2680 2685
Thr Thr Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser Ala Glu Ala
2690 2695 2700
Ser Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Met Arg Ile Ser Thr
2705 2710 2715 2720
Pro Ser Asp Gly Ser Thr Pro Leu Thr Ser Ile Leu Val Ser Thr Leu
2725 2730 2735
Pro Val Ala Ser Ser Glu Ala Ser Thr Val Ser Thr Thr Ala Val Asp
2740 2745 2750
Thr Ser Ile Pro Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr
2755 2760 2765
Thr Ala Glu Val Thr Ser Met Pro Thr Ser Thr Pro Ser Glu Thr Ser
2770 2775 2780
Thr Pro Leu Thr Ser Met Pro Val Asn His Thr Pro Val Ala Ser Ser
2785 2790 2795 2800
Glu Ala Gly Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr Pro Val
2805 2810 2815
Thr Thr Ser Thr Lys Ala Ser Ser Ser Pro Thr Thr Ala Glu Gly Ile
2820 2825 2830
Val Val Pro Ile Ser Thr Ala Ser Glu Gly Ser Thr Leu Leu Thr Ser
2835 2840 2845
Ile Pro Val Ser Thr Thr Pro Val Ala Ser Ser Glu Ala Ser Thr Leu
2850 2855 2860
Ser Thr Thr Pro Val Asp Thr Ser Ile Pro Val Thr Thr Ser Thr Glu
2865 2870 2875 2880
Gly Ser Ser Ser Pro Thr Thr Ala Glu Gly Thr Ser Met Pro Ile Ser
2885 2890 2895
Thr Pro Ser Glu Val Ser Thr Pro Leu Thr Ser Ile Leu Val Ser Thr
2900 2905 2910
Val Pro Val Ala Gly Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val
2915 2920 2925
Asp Thr Arg Thr Pro Val Thr Thr Ser Ala Glu Ala Ser Ser Ser Pro
2930 2935 2940
Thr Thr Ala Glu Gly Thr Ser Met Pro Ile Ser Thr Pro Gly Glu Arg
2945 2950 2955 2960
Arg Thr Pro Leu Thr Ser Met Ser Val Ser Thr Met Pro Val Ala Ser
2965 2970 2975
Ser Glu Ala Ser Thr Leu Ser Arg Thr Pro Ala Asp Thr Ser Thr Pro
2980 2985 2990
Val Thr Thr Ser Thr Glu Ala Ser Ser Ser Pro Thr Thr Ala Glu Gly
2995 3000 3005
Thr Gly Ile Pro Ile Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr
3010 3015 3020
Ser Ile Pro Val Ser Thr Thr Pro Val Ala Ile Pro Glu Ala Ser Thr
3025 3030 3035 3040
Leu Ser Thr Thr Pro Val Asp Ser Asn Ser Pro Val Val Thr Ser Thr
3045 3050 3055
Glu Val Ser Ser Ser Pro Thr Pro Ala Glu Gly Thr Ser Met Pro Ile
3060 3065 3070
Ser Thr Tyr Ser Glu Gly Ser Thr Pro Leu Thr Gly Val Pro Val Ser
3075 3080 3085
Thr Thr Pro Val Thr Ser Ser Ala Ile Ser Thr Leu Ser Thr Thr Pro
3090 3095 3100
Val Asp Thr Ser Thr Pro Val Thr Thr Ser Thr Glu Ala His Ser Ser
3105 3110 3115 3120
Pro Thr Thr Ser Glu Gly Thr Ser Met Pro Thr Ser Thr Pro Ser Glu
3125 3130 3135
Gly Ser Thr Pro Leu Thr Tyr Met Pro Val Ser Thr Met Leu Val Val
3140 3145 3150
Ser Ser Glu Asp Ser Thr Leu Ser Ala Thr Pro Val Asp Thr Ser Thr
3155 3160 3165
Pro Val Thr Thr Ser Thr Glu Ala Thr Ser Ser Thr Thr Ala Glu Gly
3170 3175 3180
Thr Ser Ile Pro Thr Ser Thr Pro Ser Glu Gly Met Thr Pro Leu Thr
3185 3190 3195 3200
Ser Val Pro Val Ser Asn Thr Pro Val Ala Ser Ser Glu Ala Ser Ile
3205 3210 3215
Leu Ser Thr Thr Pro Val Asp Ser Asn Thr Pro Leu Thr Thr Ser Thr
3220 3225 3230
Glu Ala Ser Ser Ser Pro Pro Thr Ala Glu Gly Thr Ser Met Pro Thr
3235 3240 3245
Ser Thr Pro Ser Glu Gly Ser Thr Pro Leu Thr Ser Met Pro Val Ser
3250 3255 3260
Thr Thr Thr Val Ala Ser Ser Glu Thr Ser Thr Leu Ser Thr Thr Pro
3265 3270 3275 3280
Ala Asp Thr Ser Thr Pro Val Thr Thr Tyr Ser Gln Ala Ser Ser Ser
3285 3290 3295
Pro Pro Ile Ala Asp Gly Thr Ser Met Pro Thr Ser Thr Tyr Ser Glu
3300 3305 3310
Gly Ser Thr Pro Leu Thr Asn Met Ser Phe Ser Thr Thr Pro Val Val
3315 3320 3325
Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Thr Ser Thr
3330 3335 3340
Pro Val Thr Thr Ser Thr Glu Ala Ser Leu Ser Pro Thr Thr Ala Glu
3345 3350 3355 3360
Gly Thr Ser Ile Pro Thr Ser Ser Pro Ser Glu Gly Thr Thr Pro Leu
3365 3370 3375
Ala Ser Met Pro Val Ser Thr Thr Pro Val Val Ser Ser Glu Val Asn
3380 3385 3390
Thr Leu Ser Thr Thr Pro Val Asp Ser Asn Thr Leu Val Thr Thr Ser
3395 3400 3405
Thr Glu Ala Ser Ser Ser Pro Thr Ile Ala Glu Gly Thr Ser Leu Pro
3410 3415 3420
Thr Ser Thr Thr Ser Glu Gly Ser Thr Pro Leu Ser Ile Met Pro Leu
3425 3430 3435 3440
Ser Thr Thr Pro Val Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr
3445 3450 3455
Pro Val Asp Thr Ser Thr Pro Val Thr Thr Ser Ser Pro Thr Asn Ser
3460 3465 3470
Ser Pro Thr Thr Ala Glu Val Thr Ser Met Pro Thr Ser Thr Ala Gly
3475 3480 3485
Glu Gly Ser Thr Pro Leu Thr Asn Met Pro Val Ser Thr Thr Pro Val
3490 3495 3500
Ala Ser Ser Glu Ala Ser Thr Leu Ser Thr Thr Pro Val Asp Ser Asn
3505 3510 3515 3520
Thr Phe Val Thr Ser Ser Ser Gln Ala Ser Ser Ser Pro Ala Thr Leu
3525 3530 3535
Gln Val Thr Thr Met Arg Met Ser Thr Pro Ser Glu Gly Ser Ser Ser
3540 3545 3550
Leu Thr Thr Met Leu Leu Ser Ser Thr Tyr Val Thr Ser Ser Glu Ala
3555 3560 3565
Ser Thr Pro Ser Thr Pro Ser Val Asp Arg Ser Thr Pro Val Thr Thr
3570 3575 3580
Ser Thr Gln Ser Asn Ser Thr Pro Thr Pro Pro Glu Val Ile Thr Leu
3585 3590 3595 3600
Pro Met Ser Thr Pro Ser Glu Val Ser Thr Pro Leu Thr Ile Met Pro
3605 3610 3615
Val Ser Thr Thr Ser Val Thr Ile Ser Glu Ala Gly Thr Ala Ser Thr
3620 3625 3630
Leu Pro Val Asp Thr Ser Thr Pro Val Ile Thr Ser Thr Gln Val Ser
3635 3640 3645
Ser Ser Pro Val Thr Pro Glu Gly Thr Thr Met Pro Ile Trp Thr Pro
3650 3655 3660
Ser Glu Gly Ser Thr Pro Leu Thr Thr Met Pro Val Ser Thr Thr Arg
3665 3670 3675 3680
Val Thr Ser Ser Glu Gly Ser Thr Leu Ser Thr Pro Ser Val Val Thr
3685 3690 3695
Ser Thr Pro Val Thr Thr Ser Thr Glu Ala Ile Ser Ser Ser Ala Thr
3700 3705 3710
Leu Asp Ser Thr Thr Met Ser Val Ser Met Pro Met Glu Ile Ser Thr
3715 3720 3725
Leu Gly Thr Thr Ile Leu Val Ser Thr Thr Pro Val Thr Arg Phe Pro
3730 3735 3740
Glu Ser Ser Thr Pro Ser Ile Pro Ser Val Tyr Thr Ser Met Ser Met
3745 3750 3755 3760
Thr Thr Ala Ser Glu Gly Ser Ser Ser Pro Thr Thr Leu Glu Gly Thr
3765 3770 3775
Thr Thr Met Pro Met Ser Thr Thr Ser Glu Arg Ser Thr Leu Leu Thr
3780 3785 3790
Thr Val Leu Ile Ser Pro Ile Ser Val Met Ser Pro Ser Glu Ala Ser
3795 3800 3805
Thr Leu Ser Thr Pro Pro Gly Asp Thr Ser Thr Pro Leu Leu Thr Ser
3810 3815 3820
Thr Lys Ala Gly Ser Phe Ser Ile Pro Ala Glu Val Thr Thr Ile Arg
3825 3830 3835 3840
Ile Ser Ile Thr Ser Glu Arg Ser Thr Pro Leu Thr Thr Leu Leu Val
3845 3850 3855
Ser Thr Thr Leu Pro Thr Ser Phe Pro Gly Ala Ser Ile Ala Ser Thr
3860 3865 3870
Pro Pro Leu Asp Thr Ser Thr Thr Phe Thr Pro Ser Thr Asp Thr Ala
3875 3880 3885
Ser Thr Pro Thr Ile Pro Val Ala Thr Thr Ile Ser Val Ser Val Ile
3890 3895 3900
Thr Glu Gly Ser Thr Pro Gly Thr Thr Ile Phe Ile Pro Ser Thr Pro
3905 3910 3915 3920
Val Thr Ser Ser Thr Ala Asp Val Phe Pro Ala Thr Thr Gly Ala Val
3925 3930 3935
Ser Thr Pro Val Ile Thr Ser Thr Glu Leu Asn Thr Pro Ser Thr Ser
3940 3945 3950
Ser Ser Ser Thr Thr Thr Ser Phe Ser Thr Thr Lys Glu Phe Thr Thr
3955 3960 3965
Pro Ala Met Thr Thr Ala Ala Pro Leu Thr Tyr Val Thr Met Ser Thr
3970 3975 3980
Ala Pro Ser Thr Pro Arg Thr Thr Ser Arg Gly Cys Thr Thr Ser Ala
3985 3990 3995 4000
Ser Thr Leu Ser Ala Thr Ser Thr Pro His Thr Ser Thr Ser Val Thr
4005 4010 4015
Thr Arg Pro Val Thr Pro Ser Ser Glu Ser Ser Arg Pro Ser Thr Ile
4020 4025 4030
Thr Ser His Thr Ile Pro Pro Thr Phe Pro Pro Ala His Ser Ser Thr
4035 4040 4045
Pro Pro Thr Thr Ser Ala Ser Ser Thr Thr Val Asn Pro Glu Ala Val
4050 4055 4060
Thr Thr Met Thr Thr Arg Thr Lys Pro Ser Thr Arg Thr Thr Ser Phe
4065 4070 4075 4080
Pro Thr Val Thr Thr Thr Ala Val Pro Thr Asn Thr Thr Ile Lys Ser
4085 4090 4095
Asn Pro Thr Ser Thr Pro Thr Val Pro Arg Thr Thr Thr Cys Phe Gly
4100 4105 4110
Asp Gly Cys Gln Asn Thr Ala Ser Arg Cys Lys Asn Gly Gly Thr Trp
4115 4120 4125
Asp Gly Leu Lys Cys Gln Cys Pro Asn Leu Tyr Tyr Gly Glu Leu Cys
4130 4135 4140
Glu Glu Val Val Ser Ser Ile Asp Ile Gly Pro Pro Glu Thr Ile Ser
4145 4150 4155 4160
Ala Gln Met Glu Leu Thr Val Thr Val Thr Ser Val Lys Phe Thr Glu
4165 4170 4175
Glu Leu Lys Asn His Ser Ser Gln Glu Phe Gln Glu Phe Lys Gln Thr
4180 4185 4190
Phe Thr Glu Gln Met Asn Ile Val Tyr Ser Gly Ile Pro Glu Tyr Val
4195 4200 4205
Gly Val Asn Ile Thr Lys Leu Arg Leu Gly Ser Val Val Val Glu His
4210 4215 4220
Asp Val Leu Leu Arg Thr Lys Tyr Thr Pro Glu Tyr Lys Thr Val Leu
4225 4230 4235 4240
Asp Asn Ala Thr Glu Val Val Lys Glu Lys Ile Thr Lys Val Thr Thr
4245 4250 4255
Gln Gln Ile Met Ile Asn Asp Ile Cys Ser Asp Met Met Cys Phe Asn
4260 4265 4270
Thr Thr Gly Thr Gln Val Gln Asn Ile Thr Val Thr Gln Tyr Asp Pro
4275 4280 4285
Glu Glu Asp Cys Arg Lys Met Ala Lys Glu Tyr Gly Asp Tyr Phe Val
4290 4295 4300
Val Glu Tyr Arg Asp Gln Lys Pro Tyr Cys Ile Ser Pro Cys Glu Pro
4305 4310 4315 4320
Gly Phe Ser Val Ser Lys Asn Cys Asn Leu Gly Lys Cys Gln Met Ser
4325 4330 4335
Leu Ser Gly Pro Gln Cys Leu Cys Val Thr Thr Glu Thr His Trp Tyr
4340 4345 4350
Ser Gly Glu Thr Cys Asn Gln Gly Thr Gln Lys Ser Leu Val Tyr Gly
4355 4360 4365
<210> 38
<211> 407
<212> PRT
<213> GUCY2C antigen
<400> 38
Ser Gln Val Ser Gln Asn Cys His Asn Gly Ser Tyr Glu Ile Ser Val
1 5 10 15
Leu Met Met Gly Asn Ser Ala Phe Ala Glu Pro Leu Lys Asn Leu Glu
20 25 30
Asp Ala Val Asn Glu Gly Leu Glu Ile Val Arg Gly Arg Leu Gln Asn
35 40 45
Ala Gly Leu Asn Val Thr Val Asn Ala Thr Phe Met Tyr Ser Asp Gly
50 55 60
Leu Ile His Asn Ser Gly Asp Cys Arg Ser Ser Thr Cys Glu Gly Leu
65 70 75 80
Asp Leu Leu Arg Lys Ile Ser Asn Ala Gln Arg Met Gly Cys Val Leu
85 90 95
Ile Gly Pro Ser Cys Thr Tyr Ser Thr Phe Gln Met Tyr Leu Asp Thr
100 105 110
Glu Leu Ser Tyr Pro Met Ile Ser Ala Gly Ser Phe Gly Leu Ser Cys
115 120 125
Asp Tyr Lys Glu Thr Leu Thr Arg Leu Met Ser Pro Ala Arg Lys Leu
130 135 140
Met Tyr Phe Leu Val Asn Phe Trp Lys Thr Asn Asp Leu Pro Phe Lys
145 150 155 160
Thr Tyr Ser Trp Ser Thr Ser Tyr Val Tyr Lys Asn Gly Thr Glu Thr
165 170 175
Glu Asp Cys Phe Trp Tyr Leu Asn Ala Leu Glu Ala Ser Val Ser Tyr
180 185 190
Phe Ser His Glu Leu Gly Phe Lys Val Val Leu Arg Gln Asp Lys Glu
195 200 205
Phe Gln Asp Ile Leu Met Asp His Asn Arg Lys Ser Asn Val Ile Ile
210 215 220
Met Cys Gly Gly Pro Glu Phe Leu Tyr Lys Leu Lys Gly Asp Arg Ala
225 230 235 240
Val Ala Glu Asp Ile Val Ile Ile Leu Val Asp Leu Phe Asn Asp Gln
245 250 255
Tyr Phe Glu Asp Asn Val Thr Ala Pro Asp Tyr Met Lys Asn Val Leu
260 265 270
Val Leu Thr Leu Ser Pro Gly Asn Ser Leu Leu Asn Ser Ser Phe Ser
275 280 285
Arg Asn Leu Ser Pro Thr Lys Arg Asp Phe Ala Leu Ala Tyr Leu Asn
290 295 300
Gly Ile Leu Leu Phe Gly His Met Leu Lys Ile Phe Leu Glu Asn Gly
305 310 315 320
Glu Asn Ile Thr Thr Pro Lys Phe Ala His Ala Phe Arg Asn Leu Thr
325 330 335
Phe Glu Gly Tyr Asp Gly Pro Val Thr Leu Asp Asp Trp Gly Asp Val
340 345 350
Asp Ser Thr Met Val Leu Leu Tyr Thr Ser Val Asp Thr Lys Lys Tyr
355 360 365
Lys Val Leu Leu Thr Tyr Asp Thr His Val Asn Lys Thr Tyr Pro Val
370 375 380
Asp Met Ser Pro Thr Phe Thr Trp Lys Asn Ser Lys Leu Pro Asn Asp
385 390 395 400
Ile Thr Gly Arg Gly Pro Gln
405
<210> 39
<211> 264
<212> PRT
<213> CD207 antigen
<400> 39
Pro Arg Phe Met Gly Thr Ile Ser Asp Val Lys Thr Asn Val Gln Leu
1 5 10 15
Leu Lys Gly Arg Val Asp Asn Ile Ser Thr Leu Asp Ser Glu Ile Lys
20 25 30
Lys Asn Ser Asp Gly Met Glu Ala Ala Gly Val Gln Ile Gln Met Val
35 40 45
Asn Glu Ser Leu Gly Tyr Val Arg Ser Gln Phe Leu Lys Leu Lys Thr
50 55 60
Ser Val Glu Lys Ala Asn Ala Gln Ile Gln Ile Leu Thr Arg Ser Trp
65 70 75 80
Glu Glu Val Ser Thr Leu Asn Ala Gln Ile Pro Glu Leu Lys Ser Asp
85 90 95
Leu Glu Lys Ala Ser Ala Leu Asn Thr Lys Ile Arg Ala Leu Gln Gly
100 105 110
Ser Leu Glu Asn Met Ser Lys Leu Leu Lys Arg Gln Asn Asp Ile Leu
115 120 125
Gln Val Val Ser Gln Gly Trp Lys Tyr Phe Lys Gly Asn Phe Tyr Tyr
130 135 140
Phe Ser Leu Ile Pro Lys Thr Trp Tyr Ser Ala Glu Gln Phe Cys Val
145 150 155 160
Ser Arg Asn Ser His Leu Thr Ser Val Thr Ser Glu Ser Glu Gln Glu
165 170 175
Phe Leu Tyr Lys Thr Ala Gly Gly Leu Ile Tyr Trp Ile Gly Leu Thr
180 185 190
Lys Ala Gly Met Glu Gly Asp Trp Ser Trp Val Asp Asp Thr Pro Phe
195 200 205
Asn Lys Val Gln Ser Val Arg Phe Trp Ile Pro Gly Glu Pro Asn Asn
210 215 220
Ala Gly Asn Asn Glu His Cys Gly Asn Ile Lys Ala Pro Ser Leu Gln
225 230 235 240
Ala Trp Asn Asp Ala Pro Cys Asp Lys Thr Phe Leu Phe Ile Cys Lys
245 250 255
Arg Pro Tyr Val Pro Ser Glu Pro
260
<210> 40
<211> 100
<212> PRT
<213> CD3 antigen
<400> 40
Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val
1 5 10 15
Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly
20 25 30
Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu
35 40 45
Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu
50 55 60
Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly
65 70 75 80
Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val
85 90 95
Cys Glu Asn Cys
100
<210> 41
<211> 371
<212> PRT
<213> CD4 antigen
<400> 41
Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys
1 5 10 15
Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His Trp Lys Asn Ser Asn
20 25 30
Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro
35 40 45
Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln
50 55 60
Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp
65 70 75 80
Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu Leu
85 90 95
Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gln Gly Gln
100 105 110
Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser Val
115 120 125
Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln Gly Gly Lys Thr Leu
130 135 140
Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly Thr Trp Thr Cys Thr
145 150 155 160
Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys Ile Asp Ile Val Val
165 170 175
Leu Ala Phe Gln Lys Ala Ser Ser Ile Val Tyr Lys Lys Glu Gly Glu
180 185 190
Gln Val Glu Phe Ser Phe Pro Leu Ala Phe Thr Val Glu Lys Leu Thr
195 200 205
Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser Ser Ser Lys
210 215 220
Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu Val Ser Val Lys Arg
225 230 235 240
Val Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys Leu Pro Leu His
245 250 255
Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr Ala Gly Ser Gly Asn Leu
260 265 270
Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys Leu His Gln Glu Val Asn
275 280 285
Leu Val Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu
290 295 300
Val Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu
305 310 315 320
Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val Leu
325 330 335
Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp Ser Gly Gln
340 345 350
Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro
355 360 365
Val Gln Pro
370
<210> 42
<211> 348
<212> PRT
<213> CD5 antigen
<400> 42
Arg Leu Ser Trp Tyr Asp Pro Asp Phe Gln Ala Arg Leu Thr Arg Ser
1 5 10 15
Asn Ser Lys Cys Gln Gly Gln Leu Glu Val Tyr Leu Lys Asp Gly Trp
20 25 30
His Met Val Cys Ser Gln Ser Trp Gly Arg Ser Ser Lys Gln Trp Glu
35 40 45
Asp Pro Ser Gln Ala Ser Lys Val Cys Gln Arg Leu Asn Cys Gly Val
50 55 60
Pro Leu Ser Leu Gly Pro Phe Leu Val Thr Tyr Thr Pro Gln Ser Ser
65 70 75 80
Ile Ile Cys Tyr Gly Gln Leu Gly Ser Phe Ser Asn Cys Ser His Ser
85 90 95
Arg Asn Asp Met Cys His Ser Leu Gly Leu Thr Cys Leu Glu Pro Gln
100 105 110
Lys Thr Thr Pro Pro Thr Thr Arg Pro Pro Pro Thr Thr Thr Pro Glu
115 120 125
Pro Thr Ala Pro Pro Arg Leu Gln Leu Val Ala Gln Ser Gly Gly Gln
130 135 140
His Cys Ala Gly Val Val Glu Phe Tyr Ser Gly Ser Leu Gly Gly Thr
145 150 155 160
Ile Ser Tyr Glu Ala Gln Asp Lys Thr Gln Asp Leu Glu Asn Phe Leu
165 170 175
Cys Asn Asn Leu Gln Cys Gly Ser Phe Leu Lys His Leu Pro Glu Thr
180 185 190
Glu Ala Gly Arg Ala Gln Asp Pro Gly Glu Pro Arg Glu His Gln Pro
195 200 205
Leu Pro Ile Gln Trp Lys Ile Gln Asn Ser Ser Cys Thr Ser Leu Glu
210 215 220
His Cys Phe Arg Lys Ile Lys Pro Gln Lys Ser Gly Arg Val Leu Ala
225 230 235 240
Leu Leu Cys Ser Gly Phe Gln Pro Lys Val Gln Ser Arg Leu Val Gly
245 250 255
Gly Ser Ser Ile Cys Glu Gly Thr Val Glu Val Arg Gln Gly Ala Gln
260 265 270
Trp Ala Ala Leu Cys Asp Ser Ser Ser Ala Arg Ser Ser Leu Arg Trp
275 280 285
Glu Glu Val Cys Arg Glu Gln Gln Cys Gly Ser Val Asn Ser Tyr Arg
290 295 300
Val Leu Asp Ala Gly Asp Pro Thr Ser Arg Gly Leu Phe Cys Pro His
305 310 315 320
Gln Lys Leu Ser Gln Cys His Glu Leu Trp Glu Arg Asn Ser Tyr Cys
325 330 335
Lys Lys Val Phe Val Thr Cys Gln Asp Pro Asn Pro
340 345
<210> 43
<211> 270
<212> PRT
<213> CD19 extracellular domain
<400> 43
Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala Val
1 5 10 15
Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu Thr
20 25 30
Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu Gly
35 40 45
Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu Phe
50 55 60
Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln Pro
65 70 75 80
Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val
85 90 95
Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly
100 105 110
Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro
115 120 125
Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg
130 135 140
Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp Ser
145 150 155 160
Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser Thr
165 170 175
Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly Pro
180 185 190
Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu Ser
195 200 205
Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu
210 215 220
Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr
225 230 235 240
Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr
245 250 255
Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly
260 265 270
<210> 44
<211> 510
<212> DNA
<213> PD-1 AE ECD aa1-170 A132L
<400> 44
atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60
ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120
ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180
gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240
gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300
cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360
tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420
gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480
aggccagccg gccagttcca aaccctggtg 510
<210> 45
<211> 170
<212> PRT
<213> PD-1 AE ECD aa1-170 A132L
<400> 45
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val
165 170
<210> 46
<211> 510
<212> DNA
<213> PD-1 WT ECD aa1-170
<400> 46
atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60
ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120
ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180
gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240
gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300
cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360
tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420
gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480
aggccagccg gccagttcca aaccctggtg 510
<210> 47
<211> 170
<212> PRT
<213> PD-1 WT ECD aa1-170
<400> 47
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val
165 170
<210> 48
<211> 63
<212> DNA
<213> IL12RB2 TM aa623-643
<400> 48
tggatggcgt ttgtggcacc aagcatttgc attgctatca tcatggtggg cattttctca 60
acg 63
<210> 49
<211> 21
<212> PRT
<213> IL12RB2 TM aa623-643
<400> 49
Trp Met Ala Phe Val Ala Pro Ser Ile Cys Ile Ala Ile Ile Met Val
1 5 10 15
Gly Ile Phe Ser Thr
20
<210> 50
<211> 63
<212> DNA
<213> PD-1 TM aa171-191
<400> 50
gttggtgtcg tgggcggcct gctgggcagc ctggtgctgc tagtctgggt cctggccgtc 60
atc 63
<210> 51
<211> 21
<212> PRT
<213> PD-1 TM aa171-191
<400> 51
Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val Trp
1 5 10 15
Val Leu Ala Val Ile
20
<210> 52
<211> 660
<212> DNA
<213> IL12RB2 ICD aa644-862
<400> 52
cattacttcc agcaaaaggt gtttgttctc ctagcagccc tcagacctca gtggtgtagc 60
agagaaattc cagatccagc aaatagcact tgcgctaaga aatatcccat tgcagaggag 120
aagacacagc tgcccttgga caggctcctg atagactggc ccacgcctga agatcctgaa 180
ccgctggtca tcagtgaagt ccttcatcaa gtgaccccag ttttcagaca tcccccctgc 240
tccaactggc cacaaaggga aaaaggaatc caaggtcatc aggcctctga gaaagacatg 300
atgcacagtg cctcaagccc accacctcca agagctctcc aagctgagag cagacaactg 360
gtggatctgt acaaggtgct ggagagcagg ggctccgacc caaagcccga aaacccagcc 420
tgtccctgga cggtgctccc agcaggtgac cttcccaccc atgatggcta cttaccctcc 480
aacatagatg acctcccctc acatgaggca cctctcgctg actctctgga agaactggag 540
cctcagcaca tctccctttc tgttttcccc tcaagttctc ttcacccact caccttctcc 600
tgtggtgata agctgactct ggatcagtta aagatgaggt gtgactccct catgctctga 660
<210> 53
<211> 219
<212> PRT
<213> IL12RB2 ICD aa644-862
<400> 53
His Tyr Phe Gln Gln Lys Val Phe Val Leu Leu Ala Ala Leu Arg Pro
1 5 10 15
Gln Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser Thr Cys Ala
20 25 30
Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro Leu Asp Arg
35 40 45
Leu Leu Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu Pro Leu Val Ile
50 55 60
Ser Glu Val Leu His Gln Val Thr Pro Val Phe Arg His Pro Pro Cys
65 70 75 80
Ser Asn Trp Pro Gln Arg Glu Lys Gly Ile Gln Gly His Gln Ala Ser
85 90 95
Glu Lys Asp Met Met His Ser Ala Ser Ser Pro Pro Pro Pro Arg Ala
100 105 110
Leu Gln Ala Glu Ser Arg Gln Leu Val Asp Leu Tyr Lys Val Leu Glu
115 120 125
Ser Arg Gly Ser Asp Pro Lys Pro Glu Asn Pro Ala Cys Pro Trp Thr
130 135 140
Val Leu Pro Ala Gly Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser
145 150 155 160
Asn Ile Asp Asp Leu Pro Ser His Glu Ala Pro Leu Ala Asp Ser Leu
165 170 175
Glu Glu Leu Glu Pro Gln His Ile Ser Leu Ser Val Phe Pro Ser Ser
180 185 190
Ser Leu His Pro Leu Thr Phe Ser Cys Gly Asp Lys Leu Thr Leu Asp
195 200 205
Gln Leu Lys Met Arg Cys Asp Ser Leu Met Leu
210 215
<210> 54
<211> 660
<212> DNA
<213> IL12RB2 DN ICD aa644-862 Y800F
<400> 54
cattacttcc agcaaaaggt gtttgttctc ctagcagccc tcagacctca gtggtgtagc 60
agagaaattc cagatccagc aaatagcact tgcgctaaga aatatcccat tgcagaggag 120
aagacacagc tgcccttgga caggctcctg atagactggc ccacgcctga agatcctgaa 180
ccgctggtca tcagtgaagt ccttcatcaa gtgaccccag ttttcagaca tcccccctgc 240
tccaactggc cacaaaggga aaaaggaatc caaggtcatc aggcctctga gaaagacatg 300
atgcacagtg cctcaagccc accacctcca agagctctcc aagctgagag cagacaactg 360
gtggatctgt acaaggtgct ggagagcagg ggctccgacc caaagcccga aaacccagcc 420
tgtccctgga cggtgctccc agcaggtgac cttcccaccc atgatggctt cttaccctcc 480
aacatagatg acctcccctc acatgaggca cctctcgctg actctctgga agaactggag 540
cctcagcaca tctccctttc tgttttcccc tcaagttctc ttcacccact caccttctcc 600
tgtggtgata agctgactct ggatcagtta aagatgaggt gtgactccct catgctctga 660
<210> 55
<211> 219
<212> PRT
<213> IL12RB2 DN ICD aa644-862 Y800F
<400> 55
His Tyr Phe Gln Gln Lys Val Phe Val Leu Leu Ala Ala Leu Arg Pro
1 5 10 15
Gln Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser Thr Cys Ala
20 25 30
Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro Leu Asp Arg
35 40 45
Leu Leu Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu Pro Leu Val Ile
50 55 60
Ser Glu Val Leu His Gln Val Thr Pro Val Phe Arg His Pro Pro Cys
65 70 75 80
Ser Asn Trp Pro Gln Arg Glu Lys Gly Ile Gln Gly His Gln Ala Ser
85 90 95
Glu Lys Asp Met Met His Ser Ala Ser Ser Pro Pro Pro Pro Arg Ala
100 105 110
Leu Gln Ala Glu Ser Arg Gln Leu Val Asp Leu Tyr Lys Val Leu Glu
115 120 125
Ser Arg Gly Ser Asp Pro Lys Pro Glu Asn Pro Ala Cys Pro Trp Thr
130 135 140
Val Leu Pro Ala Gly Asp Leu Pro Thr His Asp Gly Phe Leu Pro Ser
145 150 155 160
Asn Ile Asp Asp Leu Pro Ser His Glu Ala Pro Leu Ala Asp Ser Leu
165 170 175
Glu Glu Leu Glu Pro Gln His Ile Ser Leu Ser Val Phe Pro Ser Ser
180 185 190
Ser Leu His Pro Leu Thr Phe Ser Cys Gly Asp Lys Leu Thr Leu Asp
195 200 205
Gln Leu Lys Met Arg Cys Asp Ser Leu Met Leu
210 215
<210> 56
<211> 386
<212> PRT
<213> PD1 ECD-PD1tm-IL7RA ICD
<400> 56
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Lys
180 185 190
Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys
195 200 205
Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser
210 215 220
Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp
225 230 235 240
Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro
245 250 255
Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln
260 265 270
Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser Phe
275 280 285
Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys
290 295 300
Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser
305 310 315 320
Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly
325 330 335
Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln Ser Gly Ile
340 345 350
Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro Ile Leu Thr Ser Leu
355 360 365
Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln
370 375 380
Asn Gln
385
<210> 57
<211> 277
<212> PRT
<213> PD1 ECD-PD1tm- IL2RG ICD
<400> 57
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Glu
180 185 190
Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val
195 200 205
Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly
210 215 220
Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val
225 230 235 240
Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala
245 250 255
Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr
260 265 270
Leu Lys Pro Glu Thr
275
<210> 58
<211> 476
<212> PRT
<213> PD1 ECD-PD1tm-IL21R ICD
<400> 58
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Ser
180 185 190
Leu Lys Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val
195 200 205
Pro Ser Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly
210 215 220
Asp Phe Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu
225 230 235 240
Leu Gly Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser
245 250 255
Cys His Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu
260 265 270
Gln Glu Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser
275 280 285
Phe Trp Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu
290 295 300
Arg Asp Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu
305 310 315 320
Asp Ala Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly
325 330 335
Tyr Pro Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu
340 345 350
Glu Asp Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys
355 360 365
Val Ser Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu
370 375 380
Asp Arg Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly
385 390 395 400
Leu Pro Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala
405 410 415
Gly Ser Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe
420 425 430
Val Gly Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro
435 440 445
Gly Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile
450 455 460
Pro Pro Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser
465 470 475
<210> 59
<211> 468
<212> PRT
<213> PD1 ECD-PD1tm-GP130 ICD
<400> 59
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Asn
180 185 190
Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro Asp Pro
195 200 205
Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro Arg His
210 215 220
Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe Thr Asp
225 230 235 240
Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe Pro Glu
245 250 255
Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn Thr Glu
260 265 270
Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser Ser Arg
275 280 285
Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn Thr Ser
290 295 300
Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg His Gln
305 310 315 320
Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln Pro Leu
325 330 335
Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp His Val
340 345 350
Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys Gln Asn
355 360 365
Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu Arg Ser
370 375 380
Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu Lys Gln
385 390 395 400
Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln Met Lys
405 410 415
Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly Thr Glu
420 425 430
Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala Thr Asp
435 440 445
Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln Gly Gly
450 455 460
Tyr Met Pro Gln
465
<210> 60
<211> 224
<212> PRT
<213> PD1 ECD-CD27tm-CD27 ICD
<400> 60
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Ile Leu Val Ile Phe
145 150 155 160
Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe Leu His
165 170 175
Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro
180 185 190
Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr
195 200 205
Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro
210 215 220
<210> 61
<211> 466
<212> PRT
<213> PD1 ECD-IL2/15RBtm-IL2/15RB ICD
<400> 61
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Ile Pro Trp Leu Gly
145 150 155 160
His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val
165 170 175
Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val
180 185 190
Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser
195 200 205
Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro
210 215 220
Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu
225 230 235 240
Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp
245 250 255
Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser
260 265 270
Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu
275 280 285
Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu
290 295 300
Glu Asp Pro Asp Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro
305 310 315 320
Gln Pro Leu Gln Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe
325 330 335
Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly
340 345 350
Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu
355 360 365
Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro
370 375 380
Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe
385 390 395 400
Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro
405 410 415
Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro
420 425 430
Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr
435 440 445
Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His
450 455 460
Leu Val
465
<210> 62
<211> 375
<212> PRT
<213> PD1 ECD-IL7RAtm-IL7RA
<400> 62
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ile Leu Leu Thr
145 150 155 160
Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala
165 170 175
Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu
180 185 190
Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys
195 200 205
Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile
210 215 220
His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu
225 230 235 240
Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu
245 250 255
Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile
260 265 270
Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly
275 280 285
Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu
290 295 300
Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu
305 310 315 320
Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser
325 330 335
Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro
340 345 350
Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met
355 360 365
Ser Ser Phe Tyr Gln Asn Gln
370 375
<210> 63
<211> 262
<212> PRT
<213> PD1 ECD-Il2RGtm- IL2RG ICD
<400> 63
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Val Val Ile Ser Val
145 150 155 160
Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu
165 170 175
Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu
180 185 190
Val Thr Glu Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys
195 200 205
Gly Leu Ala Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu
210 215 220
Val Ser Glu Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly
225 230 235 240
Ala Ser Pro Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr
245 250 255
Thr Leu Lys Pro Glu Thr
260
<210> 64
<211> 461
<212> PRT
<213> PD1 ECD-IL21Rtm-IL21R ICD
<400> 64
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Gly Trp Asn Pro His
145 150 155 160
Leu Leu Leu Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp
165 170 175
Ser Leu Lys Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala
180 185 190
Val Pro Ser Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser
195 200 205
Gly Asp Phe Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu
210 215 220
Glu Leu Gly Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr
225 230 235 240
Ser Cys His Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu
245 250 255
Leu Gln Glu Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro
260 265 270
Ser Phe Trp Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu
275 280 285
Glu Arg Asp Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val
290 295 300
Leu Asp Ala Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp
305 310 315 320
Gly Tyr Pro Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly
325 330 335
Leu Glu Asp Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly
340 345 350
Cys Val Ser Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu
355 360 365
Leu Asp Arg Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly
370 375 380
Gly Leu Pro Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu
385 390 395 400
Ala Gly Ser Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly
405 410 415
Phe Val Gly Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser
420 425 430
Pro Gly Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val
435 440 445
Ile Pro Pro Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser
450 455 460
<210> 65
<211> 454
<212> PRT
<213> PD1 ECD-GP130tm-GP130 ICD
<400> 65
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Ala Ile Val Val Pro
145 150 155 160
Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys
165 170 175
Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro
180 185 190
Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro
195 200 205
Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe
210 215 220
Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe
225 230 235 240
Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn
245 250 255
Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser
260 265 270
Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn
275 280 285
Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg
290 295 300
His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln
305 310 315 320
Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp
325 330 335
His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys
340 345 350
Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu
355 360 365
Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu
370 375 380
Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln
385 390 395 400
Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly
405 410 415
Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala
420 425 430
Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln
435 440 445
Gly Gly Tyr Met Pro Gln
450
<210> 66
<211> 239
<212> PRT
<213> PD1 ECD-PD1tm-CD27 ICD
<400> 66
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Gln
180 185 190
Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro Ala
195 200 205
Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile
210 215 220
Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro
225 230 235
<210> 67
<211> 477
<212> PRT
<213> PD1 ECD-PD1tm-IL2/15RB ICD
<400> 67
Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln
1 5 10 15
Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp
20 25 30
Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp
35 40 45
Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val
50 55 60
Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala
65 70 75 80
Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
165 170 175
Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Asn
180 185 190
Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn Thr
195 200 205
Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly Gly
210 215 220
Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser
225 230 235 240
Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu Arg
245 250 255
Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu Pro
260 265 270
Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn Gln
275 280 285
Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys
290 295 300
Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu
305 310 315 320
Gly Val Ala Gly Ala Pro Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro
325 330 335
Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp
340 345 350
Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser
355 360 365
Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu Glu Arg Met Pro Pro Ser
370 375 380
Leu Gln Glu Arg Val Pro Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro
385 390 395 400
Pro Thr Pro Gly Val Pro Asp Leu Val Asp Phe Gln Pro Pro Pro Glu
405 410 415
Leu Val Leu Arg Glu Ala Gly Glu Glu Val Pro Asp Ala Gly Pro Arg
420 425 430
Glu Gly Val Ser Phe Pro Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe
435 440 445
Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser
450 455 460
Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val
465 470 475
Claims (11)
1.一种包含嵌合细胞因子受体的修饰细胞，其中所述CCR包含细胞外结构域，跨膜结构域和细胞内结构域，所述细胞外结构域结合免疫抑制剂，并且所述细胞内结构域传递细胞因子信号。
2.根据权利要求1所述的修饰的细胞，其中所述免疫抑制剂包含检查点抑制剂。
3.根据权利要求1所述的修饰的细胞，其中所述免疫抑制剂是PD-1，CTLA-4，LAG-3，TIM-3或BLTA，其中与野生型相比，PD-1，CTLA-4，LAG-3，TIM-3或BLTA的胞外结构域被修饰以增强受体-配体亲和力。
4.根据权利要求1-3中任一项所述的修饰的细胞，其中所述细胞内结构域包含IL-12R，IL-6R，IL-2R或CD27的信号转导细胞因子胞内结构域。
5.根据权利要求1-4中任一项所述的修饰的细胞，其中所述CCR是所述免疫抑制剂受体的显性失活变体，或者所述分离的核酸能够提供所述受体的显性失活变体免疫抑制剂给宿主细胞。
6.根据权利要求1-5中任一项所述的修饰的细胞，其中所述修饰的细胞包含SEQ IDNO：56-67的任一氨基酸序列。
7.根据权利要求1-6中任一项所述的分离的核酸序列或修饰的细胞，其中所述修饰的细胞包含嵌合抗原受体。
8.权利要求7的修饰的细胞，其中所述CAR包含细胞外结构域，跨膜结构域和细胞内结构域，所述细胞外结构域结合抗原。
9.权利要求8的分离的核酸序列或修饰的细胞，其中所述胞内结构域包含共刺激信号传导区，所述共刺激信号传导区包含选自CD27，CD28,4-1BB，OX40，CD30，CD40，PD-1，ICOS，淋巴细胞功能相关抗原-1(LFA-1)，CD2，CD7，LIGHT，NKG2C，B7-H3及其任何组合，其中抗原包含选自表皮生长因子受体(EGFR)，表皮生长因子受体(EGFRvIII)的变体III，人表皮生长因子受体2(HER2)，间皮素(MSLN)，前列腺特异性膜抗原(PSMA)，癌胚抗原(CEA)，二糖神经节苷脂2(GD2)，白介素-13Ra2(IL13Rα2)，磷脂酰肌醇蛋白聚糖-3(GPC3)，碳酸酐酶IX(CAIX)，L1细胞粘附分子(L1-CAM)，癌抗原125(CA125)，分化簇133(CD133)，成纤维细胞活化蛋白(FAP)，癌/睾丸抗原1B(CTAG1B)，粘蛋白1(MUC1)，叶酸受体-α(FR-α)CD19，FZD10，TSHR，PRLR，Muc17，GUCY2C，CD207，CD3，CD5，B细胞成熟抗原(BCMA)或CD4。
10.权利要求1-6中任一项的修饰的细胞，其中所述修饰的细胞包含修饰的T细胞受体(TCR)或所述分离的核酸序列包含编码所述修饰的TCR的核酸序列，其中所述TCR源自患者中自发发生的肿瘤特异性T细胞，其中所述肿瘤抗原包含CEA，gp100，MART-1，p53，MAGE-A3或NY-ESO-1，并且其中所述TCR包含TCRγ和TCRδ链或TCRα和TCRβ链或其组合。
11.一种药物组合物，其包含权利要求1-10中任一项的细胞群。
Applications Claiming Priority (2)
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| CN114807042A (zh) * | 2021-01-22 | 2022-07-29 | 南京助天中科科技发展有限公司 | 一种嵌合抗原受体改造的nk细胞及其制备方法与应用 |
| WO2024027815A1 (zh) * | 2022-08-05 | 2024-02-08 | 江苏恒瑞医药股份有限公司 | 特异性结合gucy2c和cd3的抗原结合分子及其医药用途 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109415687A (zh) * | 2016-04-07 | 2019-03-01 | 蓝鸟生物公司 | 嵌合抗原受体t细胞组合物 |
| CN112779223A (zh) * | 2019-11-08 | 2021-05-11 | 上海斯丹赛生物技术有限公司 | 偶联嵌合抗原受体细胞及其用途 |
| CN112779223B (zh) * | 2019-11-08 | 2024-06-11 | 浙江煦顼技术有限公司 | 偶联嵌合抗原受体细胞及其用途 |
| CN114807042A (zh) * | 2021-01-22 | 2022-07-29 | 南京助天中科科技发展有限公司 | 一种嵌合抗原受体改造的nk细胞及其制备方法与应用 |
| WO2024027815A1 (zh) * | 2022-08-05 | 2024-02-08 | 江苏恒瑞医药股份有限公司 | 特异性结合gucy2c和cd3的抗原结合分子及其医药用途 |
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