CN1110557C - Lag－3蛋白的可溶性多肽组分;生产方法、治疗组合物、抗独特型抗体 - Google Patents
Lag－3蛋白的可溶性多肽组分;生产方法、治疗组合物、抗独特型抗体 Download PDFInfo
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Abstract
本发明涉及一种可溶性多肽组分，它由LAG-3蛋白的4种免疫球蛋白类型胞外结构域(序列SEQ ID No.1上的氨基酸从1～159、160～239、240～330和331～412)中的至少一个的全部或部分组成，或由通过置换、添加和/或缺失一个或多个氨基酸而由上述结构域衍生的肽序列组成，它对LAG-3的配体的专一性至少等于LAG-3对其配体的专一性。
Description
本发明涉及由LAG-3膜蛋白衍生而来的可溶性物质，它可被用作免疫抑制剂和能够阻止LAG-3蛋白与MHC(主要组织相容性复合体)II型分子专一性结合的免疫刺激剂。
在WO-A91/10682中，披露了一种被称为LAG-3的蛋白质。
LAG-3蛋白是一种由NK细胞和激活的T淋巴细胞选择性地表达的蛋白质。氨基酸序列的相似性、相当的外显子/内含子结构和染色体定位表明，LAG-3与CD4相关。TRIEBEL等公开了LAG-3基因的原始特征(1)。
相应的DNA编码有498个氨基酸的I型跨膜蛋白，该蛋白带有免疫球蛋白类型的4个胞外序列。LAG-3是该免疫球蛋白超家族的一员。
其成熟蛋白包括476个氨基酸(SEQ ID No.1)，理论分子量为52KD。其胞外区有8个半胱氨酸残基和4个潜在的N-糖基化位点。Western印迹分析表明，在PHA细胞和被激活的NK细胞内的LAG-3的表观分子量为70,000。在用N-糖苷酶F处理之后，其分子量降为60kD，因而证实天然LAG-3是糖基化的。更详细的说明见WO-A91/10682。
另外，BAIXERAS等在J.Exp.Med.176，327-337中公开了他们的发现(2)：用LAG-3转染过的细胞(在其表面表达LAG-3)与表达MHC II型分子的B淋巴细胞之间的花结形成专门取决于LAG-3/MHC II型分子的相互作用。
令人惊奇的是，这种MHC II型的配体在激活的CD8+淋巴细胞(MHC I型限制性)上检测到的水平高于在CD4+淋巴细胞上的水平。在包括初级淋巴器官在内的非增生淋巴组织(即胸腺和骨髓)活体中，仅发现很少的弥漫性LAG-3+细胞(MHC II型限制性)。LAG-3+细胞见于增生淋巴小结和扁桃体，并见于被注射了大剂量IL-2的患者的外周血单核细胞(PBMC)中。
这些发现证实LAG-3是一种活化抗原，与在静息淋巴细胞亚群和其它细胞类型，特别是巨噬细胞中表达的CD4相反。
MHC包括I型和II型分子，它们是糖蛋白，向T淋巴细胞受体(TCR)呈递蛋白抗原片断。I型分子负责将主要由内源合成的蛋白衍生的肽呈递给CD8+胞毒细胞，而II型分子负责将主要源自己进入胞内的外源蛋白质，即外来途径蛋白质的肽呈递给CD4+辅助淋巴细胞。辅助T淋巴细胞调节并放大免疫响应，即为了杀死与表达“非自身”抗原，例如病毒抗原无关的细胞，就需要胞毒淋巴细胞。识别的机制涉及导致T淋巴细胞的有效活性的胞内信号。
很显然，为了起动由T(CD4+)淋巴细胞介导的免疫响应，外源抗原必须以肽的形式被特化细胞即抗原呈递细胞(APC)捕捉并内化。所产生的抗原肽在所述抗原呈递细胞表面得到再表达，并在那里与MHCII型分子结合。这种MHC II型/肽复合体能被T淋巴细胞受体专一地识别，从而激活辅助T淋巴细胞。
此外，通过重组技术创造的动物模型，使我们能够证实MHC II型分子及其配体在活体内所起的作用。
因此，缺乏MHC II型分子且几乎没有外周CD4+T淋巴细胞，而且在胸腺中仅有极少的不成熟的CD4+淋巴细胞的小鼠(3)，已被证实完全不能对依赖T细胞的抗原作出响应。
CD4-/-突变小鼠(4)的T淋巴细胞活性明显降低，但能表现出正常发育和CD8+T淋巴细胞功能，证实了CD4在子代细胞和CD4+CD8+胸腺细胞上的表达并不妨碍其发育。与正常小鼠相比，这种CD4-缺乏型小鼠有大量的CD4-CD8-细胞。
这种双负细胞仅局限于MHC II型，并能识别上述抗原。
当其感染利是曼(Leishmania)病时，这些小鼠中会出现功能性辅助T淋巴细胞的群体，尽管缺乏CD4。这些细胞局限于MHC II型，并在其被抗原激活时产生干扰素-γ。这表明T淋巴细胞及其外周功能无须依赖于CD4的功能。
现在已认识到，由MHC II型区编码的蛋白质涉及免疫识别的很多方面，包括不同的淋巴样细胞(如淋巴细胞)和抗原呈递细胞之间的相互作用。不同的观察还证明了不经过CD4进行的其它机制，这些机制参与辅助T淋巴细胞的效应子功能。
上述不同的发现突出了MHC II型及其配体在免疫系统所起的重要作用。
另外，己认识到由能够与配体结合的蛋白质的胞质外结构域和人体免疫球蛋白(Ig)链的恒定区组成的嵌合分子对于获得可溶性蛋白质和细胞受体的重要性，这种嵌合分子特别适于用作治疗剂。
因此，已证实可溶性CD4在体外能以取决于剂量的形式抑制HIV感染。
然而，采用可溶性CD4分子，特别是CD4-Ig的临床试验并未能验证其能够显著降低病毒效价。已创造出能在其血清中表达高达20μg/ml可溶性CD4的转基因小鼠。这种小鼠的免疫功能与对照小鼠没有差异。至今尚未见由CD4衍生的直接与MHC II型结合的分子的报导。这有力地表明，在体内可溶性CD4分子不与MHC II型分子相互作用。
令人惊奇的是，本发明的作者已经证实带有LAG-3蛋白的胞质外结构域的不同片段的可溶性分子能够与MHC II型分子结合，并具有免疫抑制剂作用。
由SEQ ID No.1表示的LAG-3蛋白的胞质外区包括分别从氨基酸1至159、160至239、240至330和331至412的结构域D1、D2、D3和D4。
因此，本发明的目的是一种可溶性多肽组分，它由LAG-3蛋白的4个类型免疫球蛋白胞外结构域(序列SEQ ID No.1从氨基酸1～159、160～239、240～330和331～412)中至少一个的全部或部分组成，或由通过置换、增加/或缺失一个或多个氨基酸而由这些结构域衍生而来的肽序列组成，它对LAG-3配体的专一性至少等于或大于LAG-3对其配体的专一性。
具体地讲，本发明包括具有通过已知的多型现象而由天然LAG-3序列衍生的序列的可溶性多肽组分。
这种可溶性多肽组分的特点是，它包括LAG-3上负责LAG-3与MHC II型分子的亲和性的肽区。
具体地讲，这种可溶性多肽组分包括由通过对上述结构域的一个或多个氨基酸进行置换，增加和/或缺失衍生而来的肽序列，它对LAG-3配体的专一性等于或大于LAG-3对其配体的，例如，LAG-3前两种类型免疫球蛋白的结构域，或LAG-3胞质外结构域的4种类型的免疫球蛋白结构域。
有利的是，这种可溶性多肽组分包括LAG-3蛋白的4种免疫球蛋白类型胞外结构域(序列SEQ ID No.1的氨基酸1～159、160～239、240～330和331～412)中的至少一个的全部或部分，其在序列SEQ ID No.1上的73、75和76几个位置上的精氨酸(Arg)中的一个或多个被谷氨酸(Glu)取代。
这种可溶性多肽最好包括一个环，如表1或2所示，在该环中构成基本连锁结构的原子的平均位置是从氨基酸46至77(SEQ ID No.1)，或与其相差不到5％。
另外，这种可溶性多肽组分最好还包括LAG-3的第二种类型的免疫球蛋白胞外结构域(D2)(从氨基酸160至239)。
这种可溶性多肽组分除了包括上述LAG-3的肽序列外，最好在其C-末端和/或N-末端有辅助肽序列，以便构成一种融合蛋白。“融合蛋白”一词是指能够修饰LAG-3蛋白的胞质外结构域的亚片段的理化特征的任何蛋白质的一部分。这种融合蛋白的例子包括上述LAG-3的胞质外结构域的片段，它与人体免疫球蛋白、最好是同I型gG4免疫球蛋白的重链-CH2-CH3连接区结合。
这种融合蛋白可以是二聚体或单聚体。这种融合蛋白可通过本领域技术人员已知的技术获得，例如，由Traunecker等人披露的技术(5)。
一般而言，生产这种包括与上述LAG-3的肽序列融合的免疫球蛋白区的融合蛋白的方法，是将编码与LAG-3或其衍生物相关的多肽区的cDNA片断，适当的话，在PCR扩增之后，和编码相应免疫球蛋白区的cDNA插入一个载体中，这个cDNA同编码相应的多肽区或LAG-3的衍生物的cDNA融合，以及在转染之后，在表达系统，特别是哺乳动物细胞，如仓鼠卵巢细胞中表达这些cDNA片段。
本发明的融合蛋白还可以通过裂解LAG-3/Ig缀合物而获得，这种缀合物在结构上包括一个合适的裂解位置。
本发明的目的还在于一种具有免疫抑制剂活性的治疗组合物，它包括本发明的一种可溶性多肽组分。该治疗组合物适于治疗需要免疫抑制的疾病，如自身免疫疾病。
本发明的目的还在于将抗LAG-3或上述由LAG-3衍生来的可溶性多肽组分的抗体，或这些抗体的片段，尤其是Fab、Fab′和F(ab′)2片段用于制备具有免疫刺激剂活性的治疗组合物。“免疫刺激剂”是指能够刺激能表达LAG-3的细胞(即T淋巴细胞或活化NK细胞)成熟、分化、增殖和/或功能化的一种分子实体。可将抗-LAG-3抗体用作免疫抑制患者的疫苗增强子或免疫刺激剂，例如，感染HIV的患者或者用免疫抑制物质治疗的患者，或将其用于刺激免疫系统，方法是清除表现出异常行为的自身细胞，如癌细胞。
抗-LAG-3抗体的免疫刺激剂活性是令人惊讶的，因为抗-CD4抗体具有免疫抑制剂作用。
这是抗体可以是多克隆的或是单克隆的，不过以单克隆抗体为佳。多克隆抗体可以按照已知方法，如由Benedict等披露的方法(6)制备。单克隆抗体比较理想，这是因为它是对单一表位专一的，而且所得结果有较好的再现性。生产单克隆抗体的方法是在现有技术中是众所周知的，特别值得一提的是由Kohler和Milstein公开的方法。YELTON等公开了该方法及其变型(7)。
本发明的目的还涉及抗本发明的抗体的抗独特型抗体，它具有LAG-3的内部结构，因此能够同MHC II型结合。尤其是，这种抗体可用作免疫抑制剂，例如，用作自身免疫疾病的免疫抑制剂。
本发明的治疗组合物包括上述可溶性LAG-3蛋白或抗体，以及可以药用的媒介物。这种治疗组合物可以通过常用技术配制。媒介物的形式可以根据所选择的施用途径而变化，服用途径有：口服、肠胃外施用、舌下施用、直肠或鼻腔施用。
对于用于肠胃外施用的治疗组合物而言，媒介物通常包括无菌水以及其它能够增进该治疗组合物的可溶性或其贮存能力的成分。肠胃外施用途径由静脉内注射、肌内注射或皮下注射组成。
所述治疗组合物可以是持续释放型的，特别是用于长期治疗，如自身免疫疾病的治疗。使用剂量取决于受治对象，特别是他/她的免疫系统获得所需程度的保护的能力。被使用的活性成分的精确量可由将开始治疗的开业医生很方便地加以确定。
除了可溶性LAG-3或本发明的抗体之外，本发明的治疗组合物还可以包括其它活性成分，合适的话，它通过化学键结合在LAG-3上或结合在本发明的抗体上。例如，上面提及的本发明的可溶性LAG-3蛋白可以同一种毒素，如蓖麻毒蛋白或白喉类毒素融合，能够结合到MHC II型分子上并能够杀死靶细胞，如白血病细胞和黑毒瘤细胞，或是与放射性同位素融合。
下面的实施例及附图将对本发明做更详细地说明。
例1
在有抗-LAG-3单克隆抗体的情况下活化T淋巴细胞系的增殖
所用的抗-LAG-3单克隆抗体是由Baixeras等公开的17B4(2)，于1992年7月10日保藏在CNCM，编号I-1240，以及由Huard等公开的11E3(8)。
这些抗体属于同I型gG1。对这些抗体对活化T淋巴细胞的生物学作用进行试验，活化细胞是由专一性抗原肽或由能表达LAG-3的自身抗原呈递细胞表达的MHC II型分子呈递的加工过的抗原进行刺激。
将一种被称为10H3的抗-CD48单克隆抗体用作无关的IgG1抗体(阴性对照)。
通过在PHA(植物凝集素)细胞和由EB病毒(EBV)转化的细胞系上进行的免疫荧光测定，确定抗-LAG-3抗体和抗一CD48抗体的饱和浓度。在增殖试验中，以5倍于饱和浓度的比例加入单克隆抗体。
所用的T淋巴细胞系一方面可以是由外周血淋巴细胞衍生而来的克隆154，它能抗一种类似流感血凝集(HA)片段的肽，其氨基酸序列从氨基酸306延伸至329(p20肽)；另一方面可以是由单个人体供体的外周淋巴细胞衍生而来的T淋巴细胞克隆即克隆28，它能抗白喉类毒素(DT)。与克隆154相关的抗原呈递细胞(APC)是同一供体(DR3/DR11)的由EBV转化的B淋巴细胞-T154。与克隆28相关的抗原呈递细胞是同一供体的由EBV转化的B淋巴细胞。该克隆局限于HLA DR7。
对克隆154来说，将APC(5×106)与各种剂量的p20肽一起在37℃下培养1.5小时，然后洗涤并辐射(10,000rad)。以3∶1的比例把上述细胞与克隆154细胞(0.5×105～10×105细胞/ml)一起涂布在96孔微滴定板上。对克隆28来说，相应的细胞/刺激细胞之比为1。
用丝裂霉素或辐射处理HLA DR7/EBV APC细胞，然后在有DT(残留于培养物中)的条件下加入T淋巴细胞中。克隆28细胞的最终浓度为100,000细胞/ml。
以培养2～10天的不同时间间隔加入〔3H〕胸苷(1μCi/孔)。
每个实验做三次。
结果为扣除了阴性对照(在没有免疫原的条件下与APC一起培养的T淋巴细胞)的cpm以后的平均cpm。增殖试验在96孔板上进行。在添加1μCi的胸苷并培养最后18小时以后，测定每个200μl孔里的滴定胸苷的吸收。所得结果为3次试验的平均值。标准偏差一般小于12％(在测出的cpm很低时稍高一些)。此外，合并混合培养物(克隆154/APC)上清液，通过0.22μm的膜过滤，分成多个样品并在-20℃下冷冻保存，直到滴定时使用，滴定时采用市售免疫测定试剂盒：Immunotech IL-2和INF-α滴定试剂盒、Genzyme IFN-γ试剂盒和GaymanChemicals IL-4试剂盒。
进行剂量确定研究，以建立克隆154的增殖曲线，在有或没有抗-LAG-3单克隆抗体或无关的单克隆抗体(阴性对照)的条件下使其接触各种剂量的p20专一性抗原。
16个分别进行的试验的单独结果表明，不管所加抗原的浓度如何，接近增殖峰的起始点不变，但通常观察到与抗-LAG-3单克隆抗体一起培养的T淋巴细胞的增殖明显延长。制备单克隆抗体17B4的Fab片段，并将其用于克隆154增殖的试验。被带有17B4 Fab片段的抗原(15μg/ml)激活的T淋巴细胞的增殖曲线类似于在有完整的17B4单克隆抗体(40μg/ml)的条件下培养的细胞的增殖曲线(图1)。这些结果表明，所观察到的生物学作用并不是由于由抗-LAG-3单克隆抗体的Fc区诱导的非专一性反应。
用11E3抗-LAG-3单克隆抗体获得了类似结果。
在有DT的条件下与相应的APC一起培养之后，再在有17B4单克隆抗体的条件下用抗原(破伤风类毒素10μg/ml)刺激克隆28。结果如图2所示。
在克隆28上观察到的抗-LAG-3单克隆抗体的作用(即增殖的延长)，类似于在克隆154上观察到的作用。
进行为测定对有抗-LAG-3单克隆抗体存在条件下培养的克隆154细胞进行抗原刺激后所发生的各种细胞现象而设计的各种试验。
在存在抗-LAG-3或抗CD48单克隆抗体或没有这些抗体的条件下对克隆154进行常规的抗原刺激期间收获细胞，并检测LAG-3和CD25跨膜蛋白受体的表达；在刺激以后，以不同的时间间隔收集培养物上清液样品，并检测IFN-γ、TNF-α、IL-4和IL-2的存在。
双色指示免疫荧光试验(抗-CD3单克隆抗体和抗-CD25单克隆抗体)表明，IL-2增加很少，但在抗原刺激5天后明显增加。用抗-CD3和11E3(抗-LAG-3)单克隆抗体进行的类似试验表明，从激活的当天开始LAG-3就超量表达。另外，通过与抗-LAG-3单克隆抗体一起培养还可以调节IL-2、IL-4、IFN-γ和TNF-α的分泌，由此证明在有抗-LAG-3单克隆抗体的条件下，不同的细胞现象都被改变，某些现象在刺激24小时以后就已发生。
上述结果间接地表明，LAG-3起着调节CD4+细胞的作用。抗-LAG-3单克隆抗体提高增殖、以及因此而起着免疫增强剂作用这样的事实表明，LAG-3与CD4+T淋巴细胞的“失活”有关，LAG-3在依赖抗原的刺激方面起负作用。
例2
LAG-3融合蛋白的瞬时表达
通过重组DNA技术，采用包括编码LAG-3的DNA和编码一种免疫球蛋白片段的DNA的适当载体，获得由LAG-3衍生而来的可溶性蛋白质。该瞬时表达系统由转染的Cos细胞组成。该系统有可能产生几个毫克的重组融合蛋白。重组DNA技术按Maniatis等公开的方法(22)进行。并按照生产者的推荐加以改进。
LAG-3 D1-D4 Ig和LAG-3 D1D2 Ig的构建
利用无5’-内切核酸酶活性并比较抗很高温度的Taq聚合酶，由一个包括LAG-3 cDNA的cDNA片段(FDC序列)扩增(30个循环)编码D1D2或D1-D4区的片段(Triebel等，(1))；在扩增之后于98℃温度下变性(由一台Perkin Elmer Cetus“DNA热循环仪”进行)。采用在下表中记载的特定引物。
将所得到的扩增片段(LAG-3 D1D2为739bp，LAG-3 D1-D4为1312bp)插入pBS质粒中(Stratagene)。
经XhoI和BglII消化之后制备出插入物，并将其引入载体pCDM7-CD8-IgG1(pCDM7是从由Stratagene销售的pCDM8而来)的XhoI/BamHI位点，如图3所示，把编码CD8的DNA序列换成编码LAG-3亚片段的DNA序列。所得到的表达载体带有编码D1D2或D1-D4的序列，该序列与编码人体IgG1链的-CH2-CH3连接区的DNA序列融合。
表3
用于通过PCR扩增LAG-3 DNA序列的引物
| 用于DNA扩增的引物 | 所得到的与亚片段Ig融合的编码亚片段 |
| 引物(5’)5′GCGC CTCGAGGCCCAGACCATAGGAGAG ATGT 3′偶联位点 未翻译的5’序列 翻译起点引物(3’)5′GCGC AGATCTCTCCAGACCCAGAACAGTGAGGTTATACAT 3′BglII D2结束偶联位点 | LAG-3 D1D2从前导序列到氨基酸241 |
| 引物(5’)与LAG-3 D1D2相同引物(3’)5′GCGC AGATCTACCTGGGCTAGACAGCTCTGTGAA 3′BglII D4结束偶联位点 | LAG-3 D1-D4从前导序列到氨基酸412 |
CDM7是由Seed等(10)为了在大肠杆菌(E.Coli)和真核细胞中克隆并表达DNA而研制的载体衍生而来的真核表达载体。CDM7具有如下特征：(i)一个用于在哺乳动物细胞中瞬时表达的人体巨细胞病毒启动子；(ii)一个用于能表达T抗原的哺乳动物细胞的常染色体复制的SV40病毒起点；(iii)πVX(类型Col E1)作为用于高拷贝数的质粒起点；(iv)用于在Tetamb和Ampamb大肠杆菌菌株中进行抗氨苄青霉素和四环素的SupF选择；(v)一个用于释放单链的M13复制起点；(vi)一个T7 RNA启动子；和(vii)一个用于有效克隆异源DNA的多接头。
在Cos细胞中的瞬时表达
采用一台Cellject装置(Eurogentech，Lieje，BE)，通过电穿孔(200V，1500μF，30-40msee)法用合适表达载体的30μg DNA(编码LAG-3 D1D2 Ig或LAG-3 D1-D4 Ig或CD8 Ig)转染Cos细胞(5×106)。再次将上述细胞涂布并在含有5％胎牛血清的培养基上培养。在转染6天以后收取上清液。
通过用17B4单克隆抗体进行的Western印迹分析，分析上清液以及转染细胞的细胞提取物中所得融合蛋白的合成。在用编码LAG-3D1D2 Ig或LAG-3 D1-D4 Ig的DNA转染过的细胞的上清液中，发现免疫反应性材料。
与此同时，作为采用同一表达系统和表达载体pCDM7-CD8(图3)的阴性对照，获得了一种重组CD8免疫粘附素(CD8 Ig)。
通过标准方法在蛋白质A-Sepharose上对重组蛋白LAG-3D1D2 Ig、LAG-3 D1-D4 Ig和CD8 Ig进行提纯。通过SDS-PAGE分析所得到的材料，随后进行考马斯染色或用抗-人体Ig抗体进行Western印迹分析。
例3
可溶性LAG-3亚片段的生产
为了生产大量的重组蛋白质，开发了一种由转染的哺乳动物细胞组成的稳定表达系统。宿主细胞是依赖贴壁的仓鼠卵巢(CHO)细胞，它是从缺乏二氢叶酸还原酶(DHFR)的CHO细胞中分离的，由于这种CHO细胞缺乏DHFR，其生长必需甘氨酸、嘌呤和胸苷。DHFR在核酸前体合成中的重要作用与DHFR缺陷型细胞对四氢叶酸类似物，如氨甲蝶呤(MTX)的敏感性结合在一起，具有两大优点。用带有DHFR基因的表达载体转染该细胞可以分泌重组DHFR-抗性克隆，而在含有大量MTX的选择培养基上培养该细胞会导致DHFR基因及与其相连的DNA的扩增。
LAG-3 D1、LAG-3 D1D2、LAG-3 D1-D4的构建
采用与上文所述相同的PCR方法，用下表中所列的引物扩增编码D1、D1D2或D1-D4区的DNA片段。
表4
用于通过PCR扩增LAG-3 DNA序列的引物
| 用于DNA扩增的引物 | 所得到的编码亚片段 |
| 引物(5’)5′CGCC GTCGACCGCTGCCCAGACCATAGGAGAG ATGTG 3′SalI偶联位点 未翻译的5’序列 翻译的起点引物(3’)5′GCGC GTCGACTTACATCGAGGCCTGGCCCAGGCGCAG 3′SalI D1结束偶联位点 | LAG-3 D1从前导序列到氨基酸149 |
| 引物(5’)与LAG-3 D1相同引物(3’)5′GCGC GTCGACTTAACCCAGAACAGTGAGGTTATAC 3′SalI D2结束偶联位点 | LAG-3 D1D2从前导序列到氨基酸239 |
| 引物(5’)与LAG-3 D1相同引物(3’)5′GCGC GTCGACTTAACCTGGGCTAGACAGCTCTGTG 3′SalI D4结束偶联位点 | LAG-3 D1-D4从前导序列到氨基酸412 |
用SalI消化所获得的扩增片段，并将其插入pUC18(Stratagene)的SalI位点。
验证扩增的序列，并按照Cole等所公开的方法(Biotechnology11，1014-1024，1993)将上述插入片段亚克隆到表达载体pCLH3 AXS V2DHFR hαIVS中(图4)。
该载体是一种真核表达载体，它具有表达cDNA及其在真核细胞中扩增的多种功能。它具有以下特征：(i)用于启动所感兴趣的基因转录的金属硫蛋白-1基因和聚腺苷酸化序列SV40(包括一个供体-受体剪接位点)的鼠启动子，(ii)一个用于获得cDNA高水平转录的人间插序列A，它带有用于α糖蛋白亚单位的基因的供体-受体剪接位点，(iii)带有pBR322的复制起点和一个抗氨苄青霉素的基因的pML序列，它被用于细菌扩增，和(iv)一个启动该序列转录的SV40 DHFR转录单位，用于转染子的选择和扩增。
在CHO细胞中的稳定表达
将编码LAG-3 D1、LAG-3 D1D2和LAG-3 D1-D4的表达载体用于转染CHO DUKX细胞，并在一种选择培养基上培养该细胞。合并在这种条件下能够增殖的细胞，并在含有大量MTX的培养基上培养。用17B4单克隆抗体通过Western印迹分析测定表达水平。在生物反应器中繁殖能够产生大量的由LAG-3衍生的重组可溶性分子的克隆，并通过离子交换层析和免疫亲和提纯所述由LAG-3衍生的材料。
Western印迹分析显示，在用编码LAG-3 D1、LAG-3 D1D2和LAG-3 D1-D4的表达载体转染过的细胞的上清液中，有表观分子量为15～18kD、34-36kD(双谱带)和55kD(可能为2条带)的几条带。这些免疫反应性材料的相应分子量与预期的糖基化LAG-3 D1Ig(139个氨基酸和一个推定的N-糖基化位点)、糖基化LAG-3D1D2 Ig(239个氨基酸，带有3个糖基化位点)和糖基化LAG-3 D1-D4 Ig(412个氨基酸，带有4个糖基化位点)的分子量相符合。
例4
LAG-3 Ig与表达MHC II型的细胞的专一性结合
通过间接免疫荧光测定研究所述单克隆抗体和LAG-3 D1-D4Ig的反应性。在存在LAG-3 D1-D4 Ig、CD8 Ig、一个鼠单克隆抗体(949)、与来自Coulter克隆的FITC(异硫氰酸荧光素)缀合的抗人MHC II型(DR、DP、DQ)或鼠Ig-FITC：一种与FITC缀合的无关的免疫球蛋白的条件下，于4℃温度下将靶细胞(4×105)培养30分钟。洗涤细胞并将其与同荧光素缀合的羊抗人Ig多克隆F(ab’)2或同荧光素缀合的羊抗鼠Ig多克隆抗体一起在4℃下培养30分钟(Coulter克隆)。
为了验证LAG-3/MHC II型的结合，将LAG-3 D1-D4 Ig与MHC II型阳性或阴性细胞一起培养。用抗II型单克隆抗体949或被编码LAG-3 D1-D4 Ig或CD8 Ig的DNA转染过的Cos细胞上清液处理表达MHC II型的4个B淋巴细胞系(L31、Phil EBV Raji、Sanchez和Personnaz)。用LAG-3 Ig以与抗II型单克隆抗体(阳性对照)相同的方式识别表达MHC II型分子的不同单元型的5个细胞系，而含有CD8 Ig(阴性对照)的上清液不与这些细胞系结合，正如所预料的。用上述相同的试剂处理4个MHC II型阴性细胞系(CEM、RJ、HSB2、K562)。它们都不与抗MHC II型(阴性对照)或LAG-3 D1-D4 Ig反应，这表明LAG-3 D1-D4与MHC II型分子的结合是专一性的。
其它实验是用(i)被编码人DR7或人DP4的基因转染过或作过其它处理的鼠成纤维细胞，(ii)表达MHC II型分子的鼠细胞，(iii)活化的人CD4+或CD8+细胞，和(iv)表达MHC II型分子的不同单元型的T淋巴细胞系(图8)进行的。
与CD8 Ig不同，LAG-3 D1-D4 Ig能与抗MHC II型单克隆抗体949一样有效地同所有表达MHC II型的细胞结合。LAG-3 D1-D4 Ig同试验过的所有DR和DP单元型结合，同由转染的鼠细胞表达的MHC II型分子结合，同鼠MHC II型分子以及由CD4+或CD8+T淋巴细胞表达的MHC II型分子结合。
上述结果首次证明，由MHC II型分子的配体衍生的可溶性分子能够与表达MHC II型分子的细胞结合。
类似实验表明，LAG-3 D1D2以专一性方式并以与LAG-3 D1-D4相同的效率同表达MHC II型分子的细胞结合。
LAG-3 Ig的结合活性和LAG-3 Ig的配体的细胞分布
用荧光素标记的抗人免疫球蛋白的羊血清测定这种免疫粘附素结合细胞配体的能力。
在这些实验中，首先于4℃将靶细胞与一种人单克隆抗体或一种免疫粘附素一起在含10％FCS(胎牛血清)的RPMI 1640中温育30分钟。然后，再将这些细胞与用于鼠单克隆抗体的FITC标记的羊抗鼠免疫球蛋白血清(Coulter)或者用于免疫粘附素的FITC标记的羊抗人免疫球蛋白血清(Tago)一起温育。洗涤两次后测定荧光，用一台Elite细胞计数器(Coultronics，Hialeah，EL)分析3,000个细胞。图9表示LAG-3 Ig、CD8 Ig、抗体949或抗体OKT3(抗-CD3，ATCC)的结合程度，方法是将统计的细胞数目表示为所测得荧光强度的对数的函数。
LAG-3 Ig结合到为获得HLA DR4分子的基因而转染过的鼠成纤维细胞上，而不能与未经转染的细胞结合。在相同条件下，CD8 Ig不能结合HLA DR4 +成纤维细胞。
通过免疫荧光对细胞群样品评价LAG-3Ig的配体的细胞分布。
LAG-3 Ig在包括用EB病毒转化的B细胞系(得自遗传学上无关的供体，包括DR1～DR10型的10个纯合系)在内的所有受试的阳性II型细胞上能观察到，在活化T和NK细胞上也能观察到。
图9用实例表明LAG-3 Ig与对II型抗原呈阳性的Daudi细胞的结合。
LAG-3 Ig的平均荧光强度类似于对II型抗原具专一性的抗体949所观察到的结果。与此相反，LAG-3 Ig与DR4(图9)、DR2、DR7或在鼠成纤维细胞表面表达的DPw4(未示出)的结合要弱于对抗体949观察到的情况。
在对T来源的II型抗原呈阴性的细胞系(外周血T细胞，CEM，HSB2，REX系)，对B来来源的II型抗原呈阴性的细胞系(RJ2.2.5系)或对非淋巴样来源的II型抗原呈阴性的细胞系(人系，红霉素类(erythromyoloid)来源的K562和源于黑素瘤细胞的系(未示出))上没有检测到结合。
另外，LAG-3 Ig结合异种的MHC II型分子，象由鼠淋巴瘤A20所表达的抗原，和由植物凝集素刺激的细胞所表达的猴II型分子(数据未示出)。
还用单克隆抗体17B4证实了LAG-3 Ig结合的专一性，其在细胞粘附实验中阻断LAG-3/MHC II型相互作用的能力已预先得到证实(图10) 。
在这些实验中，在将LAG-3 Ig分子与Daudi细胞进行接触之前，预先将LAG-3 Ig分子与培养基、或与17B4(1mg/ml)、或与OKT3(1mg/ml)于4℃温育30分钟。
图10表明，LAG-3 Ig与17B4预先温育抑制了与II型+细胞的结合，而对OKT3对照没有检测出抑制作用。
实施例5
IAG-3的可溶性片断对LAG-3/MHCII型相互作用的抑制
采用与可溶性片断的竞争性实验，相对于LAG-3 Ig与II型MHC分子的结合，可以直接观察到LAG-3的可溶性片断对LAG-3/MHC II型相互作用的抑制。
为证实由CHO细胞产生的可溶性LAG-3 D1D2片断是否能代替得自LAG-3的免疫粘附素的结合，进行如下试验：
将Daudi细胞与可溶性LAG-3 D1D2片断一起温育，以使这些分子结合到在Daudi细胞表面上表达的MHC II型抗原上。
第二步中，在存在单聚体形式的LAG-3 D1D4 Ig或单聚体形式的LAG-3 D1D2 Ig的条件下，温育这些细胞。
用结合到荧光素上的羊抗人IgF(ab’)2(GAH-FITC)测定衍生于LAG-3的这些免疫粘附素的结合。
对照组是未与可溶性LAG-3 D1D2片断预先温育、但与二聚体LAG-3 D1D4 Ig或单聚体LAG-3 D1D2 Ig温育过的Daudi细胞。
结果示于表5中，它表明，可溶性LAG-3 D1D2片断能够代替衍生自LAG-3的单聚体或二聚体形式的免疫粘附素。
表5
| 反应物 | 检测 | 平均荧光 | 结论 |
| --- | GAH-FITC | 0.3 | GAH不干拢 |
| 二聚体LAG-3 D1D4 Ig | GAH-FITC | 20.8 | CHO/LAG-3 D1D2的结合抑制二聚体LAG-3 D1D4 Ig的结合(58％) |
| CHO/LAG-3 D1D2，然后二聚体LAG-3 D1D4 Ig | GAH-FITC | 8.5 | |
| 单聚体LAG-3 D1D2 Ig | GAH-FITC | 62.5 | CHO/LAG-3 D1D2的结合抑制单聚体LAG-3 D1D2 Ig的结合(27％) |
| CHO/LAG-3 D1D2，然后单聚体LAG-3 D1D2 Ig | GAH-FITC | 10.9 |
这些数据证实了可溶性LAG-3D1D2片断与MHC II型分子相结合。
LAG-3/MHC II型和CD4/MHC II型相互作用的抑制
Baixeras等(2)证实了由野生型LAG-3转染的Cos细胞与由表达MHC II型分子的EBV转化的B淋巴细胞之间的花结形成。这种相互作用能被抗LAG-3和抗MHC II型的单克隆抗体抑制。
对在该文献中所披露的上述方法做如下改进：通过在与表达LAG-3的Cos细胞一起培养51Cr标记的B淋巴细胞以后计数残留的放射性的方式代替对结合在B淋巴细胞上的Cos细胞进行观察和计数(结合试验)。
研究了由LAG-3衍生的可溶性分子对LAG-3/MHC II型相互作用以及对CD4/MHC II型相互作用的可能的抑制作用。
用合适的表达载体(编码野生型LAG-3或CD4)转染Cos细胞。两天后，用胰蛋白酶处理上述Cos细胞，并以0.05×106细胞/孔的密度将其涂布在平底的12孔组织培养板上。24小时之后，将51Cr-标记的Daudi细胞(5.5×106)在该单层Cos细胞(终体积：1ml)上培养1小时。然后将靶B细胞吸走，并洗涤上述孔5～7次，轻轻滴加入1ml培养基。通过用巴斯吸管抽吸洗涤上述诸孔的边缘。用1ml PBS、10％Triton在37℃下对残留的细胞进行裂解处理15分钟。裂解产物以3000rpm的速度离心10分钟，收集100μl所得到的上清液。
在51Cr结合试验中，用LAG-3 D1-D4 Ig抑制LAG 3/MHC II型和CD4/MHC II型相互作用。同时试验人CD8 Ig和IgG1，并将其用作阴性对照。
测出LAG-3 D1-D4 Ig能显著抑制LAG-3/MHC II型的相互作用(图5A)。不过，LAG-3/MHC II型的相互作用，只能受到人CD8 Ig和IgG1的部分地、非专一性地抑制。而且，在CD4/MHC II型相互作用不受人CD8 Ig或IgG1影响的实验条件下，LAG-3 Ig被证明是CD4/MHC II型的相互作用的潜在抑制剂(图5B)。这表明LAG-3/MHC II型的相互作用弱于CD4/MHC II型的相互作用。上述结果首次揭示了可溶性分子在MHC II型与其配体的相互作用中的可能的竞争性。
例6
LAG-3 D1-D4 Ig的免疫抑制剂活性
用上面所述的增殖试验进行检验抗-LAG-3单克隆抗体活性的功能试验。
在抗原刺激以后的3天和5天(D3和D5)，LAG-3 D1-D4 Ig表现出对克隆28增殖的强烈抑制作用，而人CD8 Ig和IgG没有作用(图6)。用克隆154进行类似实验(图7)，在存在LAG-3 Ig的条件下表现出部分抑制。用抗-LAG-3单克隆抗体所做的对照有相反的效果，如在前面所观察到的。
在克隆28中，也观察到了在存在LAG-3 D1-D4 Ig的条件下培养细胞，对细胞增殖的明显抑制作用。
上述发现表明，LAG-3 D1-D4 Ig是由抗原刺激的T淋巴细胞增殖的潜在免疫抑制剂，还表明LAG-3可能起着由活化CD4+辅助T淋巴细胞诱导的再次免疫反应的“灭火剂”的作用。
LAG-3 Ig在T细胞免疫应答的负调节中的作用
为证实LAG-3的可溶形式类似于膜分子的功能，能抑制由抗原刺激的CD4 +T克隆的活化，对克隆T154进行如下试验：预先将T细胞与饱和量的LAG-3 Ig(100nM)一起温育。然后用冷RPMI洗涤细胞两次，并与10μg/ml羊抗人免疫球蛋白抗体(Tago)于4℃温育30分钟。
再洗涤两次后，将细胞重新悬浮于含10％胎牛血清的RPMI中，在加入信号之前于37℃温育2小时。为偶联(“交联”)单克隆抗体，使用浓度为10μg/ml的羊抗鼠抗体(Tago)。
图11表示一种实验，其中克隆T154已经预先与结合(“交联”)到第二反应物(对人免疫球蛋白的恒定区具有专一性的多克隆血清)上的LAG-3 Ig一起温育过。用免疫荧光法测定LAG-3 Ig与细胞的结合程度(图11A)。图11B表明，LAG-3 Ig对克隆T154增殖的抑制作用达50％以上。在相同实验条件下，对对照CD8 Ig或对没有“交联”的LAG-3 Ig没有观察到效果(未图示)。
图11C还表明，当用LAG-3 Ig来结合(“交联”)由抗原呈递B细胞表达的MHC II型分子时，观察不到效果。
把结合的(“偶联”的)抗-II型单克隆抗体对T细胞增殖的可能效果与LAG-3 Ig的效果进行对比。对结合到羊抗鼠多克隆血清上的抗体949和抗体D1.12(抗-DR)，仅观察到弱抑制(低于50％)。因此，对增殖的抑制作用是表位依赖性的，对结合II型具有专一性的LAG-3表位获得最大效果。
还用不同信号，对其它CD4 +T克隆、对碱性髓鞘蛋白的肽34-53具有专一性的克隆TDEL进行了LAG-3 Ig对T细胞增殖的影响的研究。
当用抗原刺激TDEL时(未示出)、用固定化OKT3刺激时(图13A)、用凝集素(PHA+PMA)刺激时(图13B)、用5IU/ml IL2刺激时(图13C)，观察到对增殖的抑制作用(n＝2)。对100IU/ml IL2，没有观察到抑制作用(图13D)。
总之，这些结果集中表明，LAG-3和MHC II型分子(它们每一个都是T细胞活化抗原)可能与T细胞应答失活期中涉及的效应子分子相似。此外，这些结果说明了T细胞间的相互作用在控制细胞免疫应答中的重要性。
实施例7
由LAG-3 Ig刺激细胞毒性
对两类效应子细胞研究了LAG-3 Ig在细胞毒性方面的作用：-新取到的人外周血淋巴细胞(PBL)，-SIB5系细胞(人NK细胞的克隆)。
这些细胞的细胞毒性活性是这样测定的：在培养基中有或没有LAG-3Ig的情况下，对由预先标记的靶细胞释放到培养基中的51Cr进行计数。
图14表明，SIB5对由EB病毒转化并带有MHC I型和II型抗原的人B细胞系(LAZ 388系)的细胞毒性程度，表示为加入培养物中不同反应物的函数。
共培养4小时后进行测定，效应子/靶(SIB5/LAZ 388)细胞之比为3∶1(空白栏)或1∶1(阴影栏)。
阴性对照包括培养基自身(MED)、免疫粘附素CD8 Ig和单克隆抗体17B4(抗-LAG-3)。
阳性对照包括3种不同的单克隆抗体：-抗II型DR抗原的抗体L243，-抗II型DR、DP、DQ抗原的抗体9.49，-抗人MHC I型抗原的抗体W632。
抗-HLA I型(W632)或II型(L243)抗体增加靶细胞的裂解(17B4对照则不然)。免疫粘附素LAG-3 Ig增加裂解。CD8 Ig对照没有效果。
图15表示了类似于上述试验的试验结果。其中，测定了PBL对Daudi细胞(HLA I-型)的细胞毒性，效应子/靶之比为50∶1(空白栏)和15∶1(阴影栏)。加入培养基中的反应物与第一个试验中所用的相同，只是没有抗体9.49和抗体17B4。抗体10H3是对CD45表面抗原具有专一性的同I型gG1免疫球蛋白。它被用作阴性对照。
对抗MHC I型抗原的抗体(W632)没有观察到变化。
从两个系列测定的数据可以看出，与阴性对照相比，LAG-3 Ig激活了NK细胞的细胞毒性。这一效果与就抗MHCII型分子的抗体观察的效果相类似。
序列表I-一般资料
(1)申请人：INSTITUT GUSTAVE ROUSSY/INSERM，APPLIED RESEARCH SYSTEMS ARS HOLDTNG N.V.
(2)发明名称：
由LAG-3衍生的可溶性肽组分、治疗组合物、抗LAG-3抗体的用途
(3)序列数：1II-有关序列SEQ ID NO.1的资料
序列特征
类型：核苷酸
长度：476
链型：双链
拓扑学：线性
分子类型：cDNA
生物体：homo sapiens
组织：T淋巴细胞
名称：LAG-3
序列描述：前导肽
----------------------------------------------------------------------------D1
| -22 Met-Trp-Glu-Ala-Gln-Phe-Leu-Gly-Leu-Leu-Phe-Leu-Gln-Pro-Leu- |
| Trp-Val-Ala-Pro-Val-Lys-Pro |
l
Leu-Gln-Pro-Gly-Ala-Glu-Val-Pro-Val-Val-Trp-Ala-Gln-Glu-Gly-16 Ala-Pro-Ala-Gln-Leu-Pro-Cys-Ser-Pro-Thr-Ile-Pro-Leu-Gln-Asp-31 Leu-Ser-Leu-Leu-Arg-Arg-Ala-Gly-Val-Thr-Trp-Gln-His-Gln-Pro-46 Asp-Ser-Gly-Pro-Pro-Ala-Ala-Ala-Pro-Gly-His-Pro-Leu-Ala-Pro-6l Gly-Pro-His-Pro-Ala-Ala-Pro-Ser-Ser-Trp-Gly-Pro-Arg-Pro-Arg- 76 Arg-Tyr-Thr-Val-Leu-Ser-Val-Gly-Pro-Gly-Gly-Leu-Arg-Ser-Gly-91 Arg-Leu-Pro-Leu-Gln-Pro-Arg-Val-Gln-Leu-Asp-Glu-Arg-Gly-Arg-106 Gln-Arg-Gly-Asp-Phe-Ser-Leu-Trp-Leu-Arg-Pro-Ala-Arg-Arg-Ala-121 Asp-Ala-Gly-Glu-Tyr-Arg-Ala-Ala-Val-His-Leu-Arg-Asp-Arg-Ala-136 Leu-Ser-Cys-Arg-Leu-Arg-Leu-Arg-Leu-Gly-Gln-Ala-Ser-Met 149---------------------------------------------------------------------D2
150 Thr-151 Ala-Ser-Pro-Pro-Gly-Ser-Leu-Arg-Ala-Ser-Asp-Trp-Val-Ile-Leu-166 Asn-Cys-Ser-Phe-Ser-Arg-Pro-Asp-Arg-Pro-Ala-Ser-Val-His-Trp-181 Phe-Arg-Asn-Arg-Gly-Gln-Gly-Arg-Val-Pro-Val-Arg-Glu-Ser-Pro-196 His-His-His-Leu-Ala-Glu-Ser-Phe-Leu-Phe-Leu-Pro-Gln-Val-Ser-211 Pro-Met-Asp-Ser-Gly-Pro-Trp-Gly-Cys-Ile-Leu-Thr-Tyr-Arg-Asp-226 Gly-Phe-Asn-Val-Ser-Ile-Met-Tyr-Asn-Leu-Thr-Val-Leu-Gly 239---------------------------------------------------------------------D3
240 Leu-241 Glu-Pro-Pro-Thr-Pro-Leu-Thr-Val-Tyr-Ala-Gly-Ala-Gly-Ser-Arg-256 Val-Gly-Leu-Pro-Cys-Arg-Leu-Pro-Ala-Gly-Val-Gly-Thr-Arg-Ser-271 Phe-Leu-Thr-Ala-Lys-Trp-Thr-Pro-Pro-Gly-Gly-Gly-Pro-Asp-Leu-286 Leu-Val-Thr-Gly-Asp-Asn-Gly-Asp-Phe-Thr-Leu-Arg-Leu-Glu-Asp-301 Val-Ser-Gln-Ala-Gln-Ala-Gly-Thr-Tyr-Thr-Cys-His-Ile-His-Leu-316 Gln-Glu-Gln-Gln-Leu-Asn-Ala-Thr-Val-Thr-Leu-Ala-Ile-Ile-Thr 330---------------------------------------------------------------------D4331 Val-Thr-Pro-Lys-Ser-Phe-Gly-Ser-Pro-Gly-Ser-Leu-Gly-Lys-Leu-346 Leu-Cys-Glu-Val-Thr-Pro-Val-Ser-Gly-Gln-Glu-Arg-Phe-Val-Trp-361 Ser-Ser-Leu-Asp-Thr-Pro-Ser-Gln-Arg-Ser-Phe-Ser-Gly-Pro-Trp-376 Leu-Glu-Ala-Gln-Glu-Ala-Gln-Leu-Leu-Ser-Gln-Pro-Trp-Gln-Cys- 391 Gln-Leu-Tyr-Gln-Gly-Glu-Arg-Leu-Leu-Gly-Ala-Ala-Val-Tyr-Phe-406 Thr-Glu-Leu-Ser-Ser-Pro-Gly 412------------------------------------------------------------------------跨膜
413 Ala-Gln-Arg-Ser-Gly-Arg-Ala-Pro-42l Gly-Ala-Leu-Pro-Ala-Gly-His-Leu-Leu-Leu-Phe-Leu-Thr-Leu-Gly-436 Val-Leu-Ser-Leu-Leu-Leu-Leu-Val-Thr-Gly-Ala-Phe-Gly-Phe-His-451 Leu-Trp 452------------------------------------------------------------------------胞质内
453 Arg-Arg-Gln-Trp-Arg-Pro-Arg-Arg-Phe-Ser-Ala-Leu-Glu-466 Gln-Gly-Ile-His-Pro-Arg-Arg-Leu-Arg-Ala-Arg 476
对考文献1.TRIEBEL T.et al.，1990，J.Exp.Med.171，1393-14052.BAIXERAS E.et al.，1992，J.Exp.Med.176，327-3373.COSGROVE D.et al.，1991，Cell 66，1051-10664.RAHEMTULLA A.et al.，1991，Nature 353，180-1845.TRAUNECKER A.et al.，1988，Nature 331，84-866.BENEDICT A.A.et al.，1967，Methods in Immunology1，197-306(1967)7.YELTON D.E.et al.，Ann.Rev.of Biochem.50，657-680(1981)8.HUARDB.et al.，Immunogenetics 39：2139.MANIATIS T.et al.(1982)，Molecular cloning：Alaboratory manual-Cold Spring Harbor Laboratory，New-York.10.SEED B.，1987，Nature 329，840-84211.COLE S.C.et al.，Biotechnology 11，1014-1024，199312.COLE S.C.et al.，Biotechnology 11，1014-1024，1993.
表1
5 *氨基酸的序号比序列SEQ ID No.小6HB1 37.662769316 30.430650711 76.363670349 PRO 44 h 0.1000 55HB2 36.667564392 29.027103424 77.288825989 PRO 44 h 0.1000 56CG 36.940769196 29.250995636 75.143180847 PRO 44 c2 -0.2000 57HG1 37.446662903 29.982709865 74.483322144 PRO 44 h 0.1000 58HG2 37.553295135 26.329860667 75.134750366 PRO 44 h 0.1000 59N 35.026676178 33.104183197 76.753837585 ALA 45 n -0.5000 60CA 35.034278870 34.544979095 76.400695801 ALA 45 ca 0.1200 61HN 34.452354431 32.747509003 77.536170959 ALA 45 hn 0.2800 62HA 35.105010986 34.659946442 75.298950195 ALA 45 h 0.1000 63C 36.209384918 35.322113037 77.076705933 ALA 45 c′ 0.3800 64O 36.163528442 35.637268066 78.268325806 ALA 45 o′ -0.3800 65CB 33.646369934 35.083190918 76.800727844 ALA 45 c3 -0.3000 66HB1 33.534698486 36.150661469 76.535202026 ALA 45 h 0.1000 67HB2 32.828392029 34.539138794 76.290328979 ALA 45 h 0.1000 68HB3 33.465579987 35.001335144 77.890525818 ALA 45 h 0.1000 69N 37.266757965 35.613758087 76.297294617 ALA 46 n -0.5000 70HN 37.216701508 35.231555939 75.346885681 ALA 46 hn 0.2800 71CA 38.489383698 36.310386658 76.786270142 ALA 46 ca 0.1200 72HA 38.262126923 36.871456146 77.716934204 ALA 46 h 0.1000 73C 39.058414459 37.311935425 75.727844238 ALA 46 c′ 0.3800 74O 38.922710419 37.100418091 74.516731262 ALA 46 o′ -0.3800 75CB 39.526046753 35.215301514 77.108406067 ALA 46 c3 -0.3000 76HB1 40.446556091 35.633434296 77.555480957 ALA 46 h 0.1000 77HB2 39.131206512 34.469978333 77.822120667 ALA 46 h 0.1000 78HB3 39.821903229 34.663463593 76.197715759 ALA 46 h 0.1000 79N 39.737388611 38.384365082 76.180320740 ALA 47 n -0.5000 80CA 40.295833588 39.429889679 75.275863647 ALA 47 ca 0.1200 81HN 39.717037201 38.512592316 77.196357727 ALA 47 hn 0.2800 82HA 39.901103973 39.319335938 74.245994568 ALA 47 h 0.1000 83C 41.869789124 39.413467407 75.170806885 ALA 47 c′ 0.3800 84O 42.518333435 40.166862488 75.906578064 ALA 47 o′ -0.3800 85CB 39.722030640 40.769996643 75.786285400 ALA 47 c3 -0.3000 86HB1 40.045078278 41.611721039 75.145561218 ALA 47 h 0.1000 87HB2 38.615306854 40.778987885 75.787467957 ALA 47 h 0.1000 88HB3 40.059757233 41.007873535 76.813346863 ALA 47 h 0.1000 89N 42.537422180 38.621597290 74.274406433 PRO 48 n -0.4200 90CA 44.009857178 38.718185425 74.045745850 PRO 48 ca 0.0600 91HA 44.539390564 38.855972290 75.008773804 PRO 48 h 0.1000 92CD 41.903011322 37.522361755 73.516281128 PRO 48 c2 0.0600 93HD1 41.081176758 37.871139526 72.860626221 PRO 48 h 0.1000 94HD2 41.490711212 36.761222839 74.206848145 PRO 48 h 0.1000 95C 44.448692322 39.844142914 73.044319153 PRO 48 c′ 0.3800 96O 43.693031311 40.225761414 72.144134521 PRO 48 o′ -0.3800 97CB 44.301902771 37.304885864 73.500595093 PRO 48 c2 -0.2000 98HB1 45.227554321 37.245040894 72.897834778 PRO 48 h 0.1000 99HB2 44.442562103 36.597515106 74.341529846 PRO 48 h 0.1000 100CG 43.051033020 36.925685883 72.702011108 PRO 48 c2 -0.2000 101HG1 43.084365845 37.389282227 71.696128845 PRO 48 h 0.1000 102HG2 42.948661804 35.835418701 72.555740356 PRO 48 h 0.1000 103N 45.700798035 40.324272156 73.165817261 GLY 49 n -0.5000 104CA 46.289730072 41.291931152 72.184875488 GLY 49 cg 0.0200 105HN 46.207077026 39.986907959 73.991729736 GLY 49 hn 0.2800 106HA1 45.620616913 41.460151672 71.317451477 GLY 49 h 0.1000 107HA2 46.357063293 42.283206940 72.672386169 GLY 49 h 0.1000 108C 47.682319641 40.950855255 71.600997925 GLY 49 c′ 0.3800 109O 48.560806274 41.811416626 71.601501465 GLY 49 o′ -0.3800 110N 47.842975616 39.718383789 71.091018677 HIS 50 n -0.5000 111HN 46.947166443 39.222202301 71.052452087 HIS 50 hn 0.2800 112CA 49.020114899 39.216835022 70.306198120 HIS 50 ca 0.1200 113HA 49.324539185 38.296161652 70.836235046 HIS 50 h 0.1000 114C 50.400863647 39.993576050 70.182189941 HIS 50 c′ 0.3800 115O 50.733478546 40.346022034 69.081466675 HIS 50 o′ -0.3800 116CB 48.455345154 38.673969269 68.953773499 HIS 50 c2 -0.2000 117HB1 49.238502502 38.051708221 68.481765747 HIS 50 h 0.1000 118HB2 47.639427185 37.951225281 69.144165039 HIS 50 h 0.1000 119CG 47.954322815 39.695293427 67.919425964 HIS 50 c5 0.1000 120ND1 46.759342194 40.404747009 68.035293579 HIS 50 np -0.4200 121CE1 46.825572968 41.133701324 66.873245239 HIS 50 c5 0.2700 122NE2 47.887611389 40.964004517 66.014495850 HIS 50 np -0.5000 123CD2 48.617565155 40.019775391 66.717041016 HIS 50 c5 0.0100 124HE1 46.043247223 41.852840424 66.655242920 HIS 50 h 0.1300 125HE2 48.092224121 41.403381348 65.108955383 HIS 50 hn 0.2800 126HD2 49.566501617 39.599807739 66.396347046 HIS 50 h 0.1300 127N 51.278770447 40.105480194 71.226615906 PRO 51 n -0.4200 128CA 52.667034149 40.618915558 71.077911377 PRO 51 ca 0.0600 129HA 52.723186493 41.393623352 70.287094116 PRO 51 h 0.1000 130CD 50.956447601 39.767082214 72.624496460 PRO 51 c2 0.0600 131HD1 50.920970917 38.670558929 72.747383118 PRO 51 h 0.1000 132HD2 49.988422394 40.190521240 72.947166443 PRO 51 h 0.1000 133C 53.707103729 39.478122711 70.804489136 PRO 51 c′ 0.3800 134O 53.623699188 38.391666412 71.391418457 PRO 51 o′ -0.3800 135CB 52.846694946 41.283794403 72.455955505 PRO 51 c2 -0.2000 136HB1 53.907955170 41.442169189 72.729286194 PRO 51 h 0.1000 137HB2 52.373264313 42.285846710 72.449127197 PRO 51 h 0.1000 138CG 52.105823517 40.370025635 73.440399170 PRO 51 c2 -0.2000 139HG1 52.782051086 39.565395355 73.789718628 PRO 51 h 0.1000 140HG2 51.753723145 40.912036896 74.337333679 PRO 51 h 0.1000 141N 54.704883575 39.729522705 69.933471680 LEU 52 n -0.5000 142CA 55.791530609 38.746330261 69.603820801 LEU 52 ca 0.1200 143HN 54.653743744 40.652229309 69.490356445 LEU 52 hn 0.2800 144HA 56.479202271 39.288208008 68.927917480 LEU 52 h 0.1000 145C 55.301837921 37.525024414 68.745040894 LEU 52 c′ 0.3800 146O 55.637695313 37.425930023 67.562049866 LEU 52 o′ -0.3800 147CB 56.671585083 38.316761017 70.829437256 LEU 52 c2 -0.2000 148HB1 56.036743164 37.710464478 71.502593994 LEU 52 h 0.1000 149HB2 57.445541382 37.602729797 70.487434387 LEU 52 h 0.1000 150CG 57.363307953 39.420749664 71.675102234 LEU 52 c1 -0.1000 151HG 56.617557526 40.200679779 71.926834106 LEU 52 h 0.1000 152CD1 57.875057220 38.833915710 73.004547119 LEU 52 c3 -0.3000 153HD11 58.353130341 39.601875305 73.642135620 LEU 52 h 0.1000 154HD12 57.048751831 38.400957367 73.601577759 LEU 52 h 0.1000 155HB13 58.618564606 38.028480530 72.852462769 LEU 52 h 0.1000 156CD2 58.531608582 40.085853577 70.927200317 LEU 52 c3 -0.3000 157HD21 59.028976440 40.857166290 71.545303345 LEU 52 h 0.1000 158HD22 59.309509277 39.355545044 70.634819031 LEU 52 h 0.1000 159HD23 58.192760468 40.592193604 70.005569458 LEU 52 h 0.1000 160N 54.534648895 36.601863861 69.354270935 ALA 53 n -0.5000 161CA 53.940563202 35.420303345 68.673507690 ALA 53 ca 0.1200 162HN 54.159572601 36.958141327 70.246543884 ALA 53 hn 0.2800 163HA 53.600482941 35.753322601 67.671340942 ALA 53 h 0.1000 164C 52.639778137 34.883575439 69.383995056 ALA 53 c′ 0.3800 165O 51.628326416 34.818698883 68.677047729 ALA 53 o′ -0.3800 166CB 55.008785248 34.330768585 68.423698425 ALA 53 c3 -0.3000 167HB1 54.582756042 33.460170746 67.892036438 ALA 53 h 0.1000 168HB2 55.828662872 34.711517334 67.787284851 ALA 53 h 0.1000 169HB3 55.479701996 33.961406708 69.351325989 ALA 53 h 0.1000 170N 52.555103302 34.484893799 70.698265076 PRO 54 n -0.4200 171CA 51.304950714 33.917499542 71.286506653 PRO 54 ca 0.0600 172HA 50.878768921 33.172523499 70.584587097 PRO 54 h 0.1000 173CD 53.705833435 34.435420990 71.626182556 PRO 54 c2 0.0600 174HD1 54.215991974 35.409980774 71.739936829 PRO 54 h 0.1000 175HD2 54.453365326 33.693523407 71.288909912 PRO 54 h 0.1000 176C 50.177021027 34.945980072 71.642837524 PRO 54 c′ 0.3800 177O 50.377983093 36.164409637 71.677795410 PRO 54 o′ -0.3800 178CB 51.867893219 33.173828125 72.519187927 PRO 54 c2 -0.2000 179HB1 51.135505676 33.051830292 73.340660095 PRO 54 h 0.1000 180HB2 52.181377411 32.150829315 72.232276917 PRO 54 h 0.1000 181CG 53.087123871 33.989192963 72.949317932 PRO 54 c2 -0.2000 182HG1 52.768703461 34.871643066 73.538475037 PRO 54 h 0.1000 183HG2 53.791828156 33.414070129 73.579086304 PRO 54 h 0.1000 184N 48.977436066 34.414421082 71.936706543 GLY 55 n -0.5000 185CA 47.822620392 35.225021362 72.404747009 GLY 55 cg 0.0200 186HN 48.958133698 33.389709473 71.929512024 GLY 55 hn 0.2800 187HA1 47.830284119 36.238574982 71.963676453 GLY 55 h 0.1000 188HA2 46.896526337 34.772380829 72.004432678 GLY 55 h 0.1000 189C 47.670829773 35.263648987 73.950416565 GLY 55 c′ 0.3800 190O 47.247509003 34.242198944 74.498336792 GLY 55 o′ -0.3800 191N 47.956153870 36.372848511 74.696281433 PRO 56 n -0.4200 192CA 47.830066681 36.396587372 76.179130554 PRO 56 ca 0.0600 193HA 48.225147247 35.457695007 76.619560242 PRO 56 h 0.1000 194CD 48.653686523 37.556163788 74.163940430 PRO 56 c2 0.0600 195HD1 48.108860016 38.040233612 73.332565308 PRO 56 h 0.1000 196HD2 49.652721405 37.256084442 73.799308777 PRO 56 h 0.1000 197C 46.361907959 36.604877472 76.668052673 PRO 56 c′ 0.3800 198O 45.890090942 37.732730865 76.845039368 PRO 56 o′ -0.3800 199CB 48.804897308 37.531764984 76.560951233 PRO 56 c2 -0.2000 200HB1 48.542221069 38.034294128 77.511970520 PRO 56 h 0.1000 201HB2 49.825592041 37.124542236 76.697402954 PRO 56 h 0.1000 202CG 48.782566071 38.488700867 75.368530273 PRO 56 c2 -0.2000 203HG1 47.903289795 39.158111572 75.434867859 PRO 56 h 0.1000 204HG2 49.679012299 39.133480072 75.321792603 PRO 56 h 0.1000 205N 45.650573730 35.488880157 76.896896362 HIS 57 n -0.5000 206HN 46.046119690 34.657379150 76.439048767 HIS 57 hn 0.2800 207CA 44.244419098 35.504474640 77.387596130 HIS 57 ca 0.1200 208HA 43.667560577 36.207649231 76.759368896 HIS 57 h 0.1000 209C 44.173530579 35.943927765 78.901000977 HIS 57 c′ 0.3800 210O 44.750030518 35.234142303 79.736030579 HIS 57 o′ -0.3800 211CB 43.610416412 34.095542908 77.185058594 HIS 57 c2 -0.2000 212HB1 44.270858765 33.323795319 77.623748779 HIS 57 h 0.1000 213HB2 42.689029694 34.034877777 77.796188354 HIS 57 h 0.1000 214CG 43.250400543 33.671833038 75.751731873 HIS 57 c5 0.1000 215ND1 44.116428375 33.723644257 74.666069031 HIS 57 np -0.4200 216CE1 43.325973511 33.139041901 73.713264465 HIS 57 c5 0.2700 217NE2 42.066158295 32.716590881 74.036811829 HIS 57 np -0.5000 218CD2 42.045005798 33.048488617 75.379264832 HIS 57 c5 0.0100 219HE1 43.712070465 33.001682281 72.711807251 HIS 57 h 0.1300 220HE2 41.370864868 32.225383759 73.464263916 HIS 57 hn 0.2800 221HD2 41.236827850 32.815635681 76.056380127 HIS 57 h 0.1300 222N 43.513858795 37.068405151 79.318038940 PRO 58 n -0.4200 223CA 43.593978882 37.569736481 80.719970703 PRO 58 ca 0.0600 224HA 44.657642365 37.630668640 81.026939392 PRO 58 h 0.1000 225CD 42.833599091 38.010932922 78.407653809 PRO 58 c2 0.0600 226HD1 42.093353271 37.516807556 77.751007080 PRO 58 h 0.1000 227HD2 43.569110870 38.527889252 77.758674622 PRO 58 h 0.1000 228C 42.805988312 36.695770264 81.747009277 PRO 58 c′ 0.3800 229O 41.569808960 36.693984985 81.769706726 PRO 58 o′ -0.3800 230CB 43.072513580 39.016571045 80.579101563 PRO 58 c2 -0.2000 231HB1 42.559799194 39.392253876 81.485702515 PRO 58 h 0.1000 232HB2 43.920654297 39.706352234 80.398040771 PRO 58 h 0.1000 233CG 42.156440735 38.999496460 79.353309631 PRO 58 c2 -0.2000 234HG1 41.151851654 38.629653931 79.634307861 PRO 58 h 0.1000 235HG2 42.023620605 39.998752594 78.896965027 PRO 58 h 0.1000 236N 43.540618896 35.961261749 82.605430603 ALA 59 n -0.5000 237HN 44.537330627 35.932937622 82.365699768 ALA 59 hn 0.2800 238CA 42.949653625 34.946208954 83.526855469 ALA 59 ca 0.1200 239HA 42.197692871 34.355514526 82.965911865 ALA 59 h 0.1000 240C 42.208984375 35.488883972 84.803985596 ALA 59 c′ 0.3800 241O 42.496433258 35.127353668 85.948333740 ALA 59 o′ -0.3800 242CB 44.107444763 33.975612640 83.839767456 ALA 59 c3 -0.3000 243HB1 43.761249542 33.130538940 84.463287354 ALA 59 h 0.1000 244HB2 44.544620514 33.531585693 82.924507141 ALA 59 h 0.1000 245HB3 44.925910950 34.467308044 84.399597168 ALA 59 h 0.1000 246N 41.192062378 36.323741913 84.559051514 ALA 60 n -0.5000 247HN 41.124549866 36.554992676 83.555343628 ALA 60 hn 0.2800 248CA 40.203086853 36.790748596 85.562988281 ALA 60 ca 0.1200 249HA 40.023948669 35.965785980 86.283721924 ALA 60 h 0.1000 250C 38.823528290 37.027305603 84.846977234 ALA 60 c′ 0.3800 251O 37.897804260 36.283885956 85.186340332 ALA 60 o′ -0.3800 252CB 40.756233215 37.971691132 86.389617920 ALA 60 c3 -0.3000 253H81 40.004146576 38.357006073 87.102043152 ALA 60 h 0.1000 254HB2 41.631908417 37.656120300 86.987319946 ALA 60 h 0.1000 255HB3 41.090072632 38.818138123 85.765472412 ALA 60 h 0.1000 256N 38.616340637 37.921970367 83.823921204 PRO 61 n -0.4200 257CA 37.403762817 37.874496460 82.948486328 PRO 61 ca 0.0600 258HA 36.491020203 37.824863434 83.573593140 PRO 61 h 0.1000 259CD 39.556003571 39.003845215 83.459777832 PRO 61 c2 0.0600 260HD1 40.594814301 38.658184052 83.304046631 PRO 61 h 0.1000 261HD2 39.571029663 39.778694153 84.250648499 PRO 61 h 0.1000 262C 37.275394440 36.712963104 81.892074585 PRO 61 c′ 0.3800 263O 36.266387939 36.670307159 81.183494568 PRO 61 o′ -0.3800 264CB 37.473857880 39.266963959 82.286987305 PRO 61 c2 -0.2000 265HB1 36.949714661 39.321308136 81.312759399 PRO 61 h 0.1000 266HB2 36.985511780 40.017913818 82.938987732 PRO 61 h 0.1000 267CG 38.968631744 39.571613312 82.168632507 PRO 61 c2 -0.2000 268HG1 39.397396088 39.046298981 81.292793274 PRO 61 h 0.1000 269HG2 39.180202484 40.649600983 82.039207458 PRO 61 h 0.1000 270N 38.242324829 35.777751923 81.785034180 SER 62 n -0.5000 271CA 38.186004639 34.624855042 80.844123840 SER 62 ca 0.1200 272HN 39.035541534 35.927360535 82.416992188 SER 62 hn 0.2800 273HA 37.921787262 35.006183624 79.841125488 SER 62 h 0.1000 274C 37.145660400 33.532897949 81.261909485 SER 62 c′ 0.3800 275O 37.466091156 32.626064301 82.041137695 SER 62 o′ -0.3800 276CB 39.620265961 34.048240662 80.725196838 SER 62 c2 -0.1700 277HB1 39.660293579 33.306644440 79.904281616 SER 62 h 0.1000 278HB2 40.349685669 34.832687378 80.442802429 SER 62 h 0.1000 279OG 40.032703400 33.414188385 81.938880920 SER 62 oh -0.3800 280HG 39.252223969 32.931293488 82.256263733 SER 62 ho 0.3500 281N 35.902244568 33.647918701 80.764541626 SER 63 n -0.5000 282CA 34.747528076 32.874465942 81.297317505 SER 63 ca 0.1200 283HN 35.768447876 34.503852844 80.205200195 SER 63 hn 0.2800 284HA 35.064254761 32.265518188 82.170570374 SER 63 h 0.1000 285C 34.106758118 31.936998367 80.231674194 SER 63 c′ 0.3800 286O 33.716896057 32.367130280 79.142120361 SER 63 o′ -0.3800 287CB 33.716815948 33.889484406 81.843544006 SER 63 c2 -0.1700 288HB1 34.199871063 34.571384430 82.572105408 SER 63 h 0.1000 289HB2 33.328830719 34.543502808 81.036796570 SER 63 h 0.1000 290OG 32.634590149 33.222091675 82.496467590 SER 63 oh -0.3800 291HG 32.159793854 32.710407257 81.832328796 SER 63 ho 0.3500 292N 33.914913177 30.658897400 80.588378906 TRP 64 n -0.5000 293CA 33.112319946 29.694124222 79.783546448 TRP 64 ca 0.1200 294HN 34.221500397 30.422899246 81.538955688 TRP 64 hn 0.2800 295HA 33.404731750 29.812168121 78.721931458 TRP 64 h 0.1000 296C 31.573041916 29.961977005 79.883049011 TRP 64 c′ 0.3800 297O 30.996980667 29.940891266 80.975959778 TRP 64 o′ -0.3800 298CB 33.525466919 26.235471725 80.142707825 TRP 64 c2 -0.2000 299HB1 32.950366974 27.534402847 79.508041382 TRP 64 h 0.1000 300HB2 34.571674347 28.078489304 79.816658020 TRP 64 h 0.1000 301CG 33.405326843 27.783784866 81.611763000 TRP 64 c5 0.0000 302CD1 32.267101288 27.214570999 82.221214294 TRP 64 c5 0.0100 303NE1 32.481933594 26.953943253 83.590408325 TRP 64 np -0.5000 304CE2 33.781982422 27.378627777 83.812339783 TRP 64 c5 0.1100 305CD2 34.355152130 27.881061554 82.617965698 TRP 64 c5 0.0000 306HD1 31.332025528 27.036033630 81.708480835 TRP 64 h 0.1000 307HE1 31.820940018 26.578481674 84.279945374 TRP 64 hn 0.2800 308CE3 35681034088 28.394184113 82.615905762 TRP 64 cp -0.1000 309HE3 36.128986359 28.784986496 81.714393616 TRP 64 h 0.1000 310CZ3 36.396430969 28.387191772 83.815704346 TRP 64 cp -0.1000 311HZ3 37.405311584 28.776082993 83.832160950 TRP 64 h 0.1000 312CH2 35.830604553 27.888952255 84.994010925 TRP 64 cp -0.1000 313HH2 36.410472870 27.896844864 85.906951904 TRP 64 h 0.1000 314CZ2 34.527233124 27.382776260 85.014289856 TRP 64 cp -0.1000 315HZ2 34.097515106 27.006088257 85.929389954 TRP 64 h 0.1000 316N 30.921329498 30.232547760 78.740600586 GLY 65 n -0.5000 317CA 29.460748672 30.504768372 78.692192078 GLY 65 cg 0.0200 318HN 31.520374298 30.302478790 77.901519775 GLY 65 hn 0.2800 319HA1 29.073087692 30.896234512 79.650825500 GLY 65 h 0.1000 320HA2 29.288171768 31.333106995 77.981094360 GLY 65 h 0.1000 321C 28.633579254 29.293350220 78.197364807 GLY 65 c′ 0.3800 322O 28.566907883 29.137302399 76.975486755 GLY 65 o′ -0.3800 323N 27.989013672 28.429246902 79.038352966 PRO 66 n -0.4200 324CA 27.282257080 27.212890625 78.546752930 PRO 66 ca 0.0600 325HA 27.989650726 26.634012222 77.917152405 PRO 66 h 0.1000 326CD 28.016592026 28.532529831 80.511337280 PRO 66 c2 0.0600 327HD1 27.731332779 29.536006927 80.880874634 PRO 66 h 0.1000 328HD2 29.027824402 28.301166534 80.897590637 PRO 66 h 0.1000 329C 25.977466583 27.479314804 77.725341797 PRO 66 c′ 0.3800 330O 25.217950821 28.417282104 77.982574463 PRO 66 o′ -0.3800 331CB 27.045602798 26.422634125 79.851196289 PRO 66 c2 -0.2000 332HB1 26.132562637 25.797079086 79.827728271 PRO 66 h 0.1000 333HB2 27.890687943 25.729280472 80.029365540 PRO 66 h 0.1000 334CG 27.003501892 27.477243423 80.958015442 PRO 66 c2 -0.2000 335HG1 25.990892410 27.921483994 81.014678955 PRO 66 h 0.1000 336HG2 27.232566833 27.061700821 81.956459045 PRO 66 h 0.1000 337N 25.734319687 26.626403809 76.719802856 ARG+ 67 n -0.5000 338CA 24.603988647 26.793025970 75.767257690 ARG+ 67 ca 0.1200 339HN 26.386735916 25.841371536 76.649803162 ARG+ 67 hn 0.2800 340HA 24.496238708 27.874828339 75.561668396 ARG+ 67 h 0.1000 341C 23.227464676 26.224872589 76.267372131 ARG+ 67 c′ 0.3800 342O 23.178310394 25.034952164 76.603759766 ARG+ 67 o′ -0.3800 343CB 24.990058899 26.165229797 74.398826599 ARG+ 67 c2 -0.2000 344HB1 24.135663986 26.318639755 73.709175110 ARG+ 67 h 0.1100 345HB2 25.787433624 26.779323578 73.940032959 ARG+ 67 h 0.1100 346CG 25.439929962 24.676465988 74.361564636 ARG+ 67 c2 -0.2000 347HG1 26.546255112 24.646316528 74.415458679 ARG+ 67 h 0.1300 348HG2 25.092346191 24.131168365 75.261718750 ARG+ 67 h 0.1300 349CD 24.934387207 23.941221237 73.112297058 ARG+ 67 c2 -0.0900 350HD1 23.838283539 23.774566650 73.188652039 ARG+ 67 h 0.1300 351HD3 25.070211411 24.585262299 72.220893860 ARG+ 67 h 0.1300 352NE 25.665744781 22.657058716 72.968780518 ARG+ 67 n1 -0.5000 353HE 26.251846313 22.313375473 73.731925964 ARG+ 67 hn 0.3600 354CZ 25.689014435 21.902687073 71.871635437 ARG+ 67 cr 0.4500 355NH1 26.493299484 20.879484177 71.859733582 ARG+ 67 n2 -0.5000 356HH11 27.072675705 20.740955353 72.690315247 ARG+ 67 hn 0.3600 357HH12 26.520929337 20.319580078 71.006805420 ARG+ 67 hn 0.3600 358NH2 24.956668854 22.117029190 70.805320740 ARG+ 67 n2 -0.5000 359HH21 25.030595779 21.456792831 70.033142090 ARG+ 67 hn 0.3600 360HH22 24.266489029 22.916227341 70.850784302 ARG+ 67 hn 0.3600 361N 22.080270767 26.971176147 76.244255066 PRO 68 n -0.4200 362CA 20.743734360 26.358839035 76.485237122 PRO 68 ca 0.0600 363HA 20.817556381 25.605155945 77.294357300 PRO 68 h 0.1000 364CD 22.076143265 28.448001862 76.342918396 PRO 68 c2 0.0600 365HD1 22.539228439 28.949869156 75.469612122 PRO 68 h 0.1000 366HD2 22.632146835 28.776126862 77.244499207 PRO 68 h 0.1000 367C 20.182382584 25.586324692 75.240180969 PRO 68 c′ 0.3800 368O 20.420539856 24.381649017 75.139877319 PRO 68 o′ -0.3800 369CB 19.948141098 27.549791336 77.062515259 PRO 68 c2 -0.2000 370HB1 18.858430862 27.490163803 76.877128601 PRO 68 h 0.1000 371HB2 20.066789627 27.567596436 78.163002014 PRO 68 h 0.1000 372CG 20.592071533 28.804227829 76.467758179 PRO 68 c2 -0.2000 373HG1 20.158363342 29.031393051 75.478172302 PRO 68 h 0.1000 374HG2 20.428398132 29.704229355 77.092338562 PRO 68 h 0.1000 375N 19.458055496 26.229068756 74.300010681 ARG+ 69 n -0.5000 376CA 18.893756866 25.542047501 73.096710205 ARG+ 69 ca 0.1200 377HN 19.221296310 27.198928833 74.529014587 ARG+ 69 hn 0.2800 378HA 19.667610168 24.872461319 72.660797119 ARG+ 69 h 0.1000 379C 18.514461517 26.608341217 72.012504578 ARG+ 69 c′ 0.3800 380O 17.383218765 27.099323273 72.036094666 ARG+ 69 o′ -0.3800 381CB 17.681777954 24.654710770 73.539657593 ARG+ 69 c2 -0.2000 382HB1 18.011884689 23.935596466 74.315147400 ARG+ 69 h 0.1100 383HB2 16.954420090 25.310214996 74.061965942 ARG+ 69 h 0.1100 384CG 16.946891785 23.862810135 72.427490234 ARG+ 69 c2 -0.2000 385HG1 16.649517059 24.551628113 71.612152100 ARG+ 69 h 0.1300 386HG2 17.638776779 23.127803802 71.965805054 ARG+ 69 h 0.1300 387CD 15.688191414 23.166488647 72.975959778 ARG+ 69 c2 -0.0900 388HD1 15.980383873 22.365550995 73.686569214 ARG+ 69 h 0.1300 389HD2 15.090404510 23.898859024 73.554351807 ARG+ 69 h 0.1300 390NE 14.889810562 22.612804413 71.848815918 ARG+ 69 n1 -0.5000 391HE 15.272388458 22.616128922 70.898582458 ARG+ 69 hn 0.3600 392CZ 13.644455910 22.143890381 71.942466736 ARG+ 69 cr 0.4500 393NH1 13.048615456 21.755460739 70.850708008 ARG+ 69 n2 -0.5000 394HH11 13.576630592 21.832328796 69.979103088 ARG+ 69 hn 0.3600 395HH12 12.090744019 21.411947250 70.936080933 ARG+ 69 hn 0.3600 396HH2 12.989639282 22.056533813 73.074882507 ARG+ 69 n2 -0.5000 397HH21 12.033639908 21.696094513 73.066261292 ARG+ 69 hn 0.3600 398HH22 13.529273987 22.372974396 73.883934021 ARG+ 69 hn 0.3600 399N 19.436628342 26.928932190 71.074501038 ARG+ 70 n -0.5000 400CA 19.223009109 27.811206818 69.878326416 ARG4 70 ca 0.1200 401HN 20.357131958 26.451332092 71.165985107 ARG+ 70 hn 0.2800 402HA 19.087514877 27.065124512 69.071128845 ARG+ 70 h 0.1000 403C 20.512538910 28.575149536 69.398536682 ARG+ 70 c′ 0.3800 404O 20.872812271 28.468791962 68.228363037 ARG4 70 o′ -0.3800 405CB 17.935552597 28.697717667 69.732887268 ARG+ 70 c2 -0.2000 406HB1 17.889257431 29.085472107 68.694030762 ARG+ 70 h 0.1100 407HB2 17.053388596 28.033058167 69.807891846 ARG+ 70 h 0.1100 408CG 17.775762558 29.883768082 70.717842102 ARG+ 70 c2 -0.2000 409HG1 18.004568100 29.546030045 71.747383118 ARG+ 70 h 0.1300 410HG2 18.540782928 30.651863098 70.495643616 ARG+ 70 h 0.1300 411CD 16.368116379 30.502414703 70.693214417 ARG+ 70 c2 -0.0900 412HD1 16.095691681 30.812221527 69.664886475 ARG+ 70 h 0.1300 413HD2 15.630161285 29.711160660 70.937812805 ARG+ 70 h 0.1300 414NE 16.255954742 31.590759277 71.711013794 ARG+ 70 n1 -0.5000 415HE 16.253637314 31.350564957 72.706428528 ARG+ 70 hn 0.3600 416CZ 16.144437790 32.900745392 71.464561462 ARG+ 70 cr 0.4500 417NH1 16.055492401 33.712890625 72.481109619 ARG+ 70 n2 -0.5000 418HH11 16.071464539 33.294216156 73.413330078 ARG+ 70 hn 0.3600 419HH12 15.975571632 34.708621979 72.277374268 ARG+ 70 hn 0.3600 420NH2 16.120351791 33.420074463 70.259872437 ARG+ 70 n2 -0.5000 421HH21 16.025018692 34.432674408 70.167800903 ARG+ 70 hn 0.3600 422HH22 16.187112808 32.736862183 69.505996704 ARG+ 70 hn 0.3600 423N 21.115812302 29.562902451 70.071769714 TYRC 71 n -0.5000 424HN 21.515977859 30.157514572 69.338050842 TYRC 71 hn 0.2800 425CA 22.034273148 29.314456940 71.218978882 TYRC 71 ca 0.1200 426HA 22.671009064 28.444923401 70.976676941 TYRC 71 h 0.1000 427C 21.352920532 28.953493118 72.563385010 TYRC 71 c′ 0.4100 428OXT 20.392858505 29.553161621 73.048652649 TYRC 71 o′ -0.3800 4290 21.928325653 27.853042603 73.145797729 TYRC 71 oh -0.3800 430HO 21.429273605 27.558662415 73.909782410 TYRC 71 ho 0.3500 431CB 22.969152451 30.555358887 71.361984253 TYRC 71 c2 -0.2000 432HB1 22.368267059 31.487627029 71.355415344 TYRC 71 h 0.1000 433HB2 23.401992798 30.559690475 72.381835938 TYRC 71 h 0.1000 434CG 24.144546509 30.666173935 70.352111816 TYRC 71 cp 0.0000 435CD1 23.927837372 31.126276016 69.044418335 TYRC 71 cp -0.1000 436HD1 22.944635391 31.424867630 68.717597961 TYRC 71 h 0.1000 437CE1 24.987041473 31.212265015 68.143028259 TYRC 71 cp -0.1000 438HE1 24.819047928 31.555503845 67.131973267 TYRC 71 h 0.1000 439CZ 26.273118973 30.861675262 68.542274475 TYRC 71 cp 0.0300 440OH 27.314481735 30.957365036 67.652267456 TYRC 71 oh -0.3800 441H 26.980180740 31.236070633 66.796859741 TYRC 71 ho 0.3500 442CE2 26.504697800 30.422637939 69.841377258 TYRC 71 cp -0.1000 443HE2 27.503232956 30.148866653 70.140815735 TYRC 71 h 0.1000 444CD2 25.447391510 30.325349808 70.743820190 TYRC 71 cp -0.1000 445HD2 25.652494431 29.968608856 71.745338440 TYRC 71 h 0.1000 430
表2
* 氨基酸的序号比序列SEQ ID No.小6HB2 36.733722687 29.095384598 77.233831069 PRO 44 h 0.1000 54CG 37.005489349 29.369808197 75.152616408 PRO 44 c2 -0.2000 55HG1 37.502185822 30.119005203 74.504158020 PRO 44 h 0.1000 56HG2 37.625408173 28.451385498 75.115615645 PRO 44 h 0.1000 57N 35.047088623 33.173435211 76.816978455 ALA 45 n -0.5000 58CA 35.011333466 34.609920502 76.449216750 ALA 45 ca 0.1200 59HN 34.471405029 32.798248291 77.590728760 ALA 45 hn 0.2800 60HA 35.065380096 34.699813843 75.343650818 ALA 45 h 0.1000 61C 36.187728882 35.414947510 77.090148926 ALA 45 c′ 0.3800 62O 36.133388519 35.819305420 78.255142212 ALA 45 o′ -0.3800 63CB 33.615478516 35.135440826 76.831176758 ALA 45 c3 -0.3000 64HB1 33.490375519 36.187480927 76.517280579 ALA 45 h 0.1000 65HB2 32.811222076 34.556259155 76.338432312 ALA 45 h 0.1000 66HB3 33.433517456 35.098365784 77.922439575 ALA 45 h 0.1000 67N 37.264499664 35.613868713 76.306388855 ALA 46 n -0.5000 68HN 37.248832703 35.072978973 75.433502197 ALA 46 hn 0.2800 69CA 38.503662109 36.298694611 76.764076233 ALA 46 ca 0.1200 70HA 38.303600311 36.883266449 77.688095093 ALA 46 h 0.1000 71C 39.082061768 37.273509979 75.687866211 ALA 46 c′ 0.3800 72O 38.951850891 37.052509308 74.481193542 ALA 46 o′ -0.3800 73CB 39.509185791 35.179004669 77.103065491 ALA 46 c3 -0.3000 74HB1 40.441535950 35.582756042 77.535072327 ALA 46 h 0.1000 75HB2 39.106670380 34.460605621 77.839447021 ALA 46 h 0.1000 76HB3 39.780502319 34.597728729 76.205062866 ALA 46 h 0.1000 77N 39.768814087 38.344066620 76.133750916 ALA 47 n -0.5000 78CA 40.322643280 39.391151428 75.225708008 ALA 47 ca 0.1200 79HN 39.783836365 38.455593109 77.149337769 ALA 47 hn 0.2800 80HA 39.932807922 39.265201569 74.196365356 ALA 47 h 0.1000 81C 41.900882721 39.401374817 75.126907349 ALA 47 c′ 0.3800 82O 42.538444519 40.171451569 75.854652405 ALA 47 o′ -0.3800 83CB 39.728843689 40.731719971 75.714279175 ALA 47 c3 -0.3000 84HB1 40.043342590 41.567916870 75.059875488 ALA 47 h 0.1000 85HB2 38.621978760 40.726497650 75.711242676 ALA 47 h 0.1000 86HB3 40.062076569 40.987442017 76.739311218 ALA 47 h 0.1000 87N 42.578651428 38.603939056 74.242019653 PRO 48 n -0.4200 88CA 44.052474976 38.702857971 74.013595581 PRO 48 ca 0.0600 89HA 44.576034546 38.850185394 74.977058411 PRO 48 h 0.1000 90CD 41.956359863 37.474979401 73.520225525 PRO 48 c2 0.0600 91HD1 41.114963531 37.788272858 72.872795105 PRO 48 h 0.1000 92HD2 41.576156616 36.723918915 74.239135742 PRO 48 h 0.1000 93C 44.492458344 39.820354462 73.002609253 PRO 48 c′ 0.3800 94O 43.782276154 40.131282806 72.040626526 PRO 48 o′ -0.3800 95CB 44.356296539 37.289741516 73.479736328 PRO 48 c2 -0.2000 96HB1 45.273612976 37.234390259 72.865592957 PRO 48 h 0.1000 97HR2 44.513816833 36.591526031 74.322021484 PRO 48 h 0.1000 98CG 43.102409363 36.884414673 72.700988770 PRO 48 c2 -0.2000 99HG1 43.119277954 37.331111908 71.685241699 PRO 48 h 0.1000 100HG2 43.010280609 35.788948059 72.572326660 PRO 48 h 0.1000 101N 45.709655762 40.366821289 73.185493469 GLY 49 n -0.5000 102CA 46.317604065 41.332912445 72.214889526 GLY 49 cg 0.0200 103HN 46.169986725 40.089691162 74.058357239 GLY 49 hn 0.2800 104BA1 45.654991150 41.537052155 71.351181030 GLY 49 h 0.1000 105HA2 46.406318665 42.313266754 72.719123840 GLY 49 h 0.1000 106C 47.710880280 40.963481903 71.654037476 GLY 49 c′ 0.3800 107O 48.630664825 41.772521973 71.754951477 GLY 49 o′ -0.3800 108N 47.830738068 39.763301849 71.063682556 HIS 50 n -0.5000 109HN 46.918842316 39.310573578 70.943237305 HIS 50 hn 0.2800 110CA 49.045799255 39.210880280 70.375061035 HIS 50 ca 0.1200 111HA 49.315334320 38.320884705 70.972137451 HIS 50 h 0.1000 112C 50.433021545 39.981941223 70.267852783 HIS 50 c′ 0.3800 113O 50.773132324 40.456230164 69.178672791 HIS 50 o′ -0.3800 114CB 48.558776855 36.590164165 69.024101257 HIS 50 c2 -0.2000 115HB1 49.390335083 38.009521464 66.577232361 HIS 50 h 0.1000 116HB2 47.792594910 37.817192078 69.227310181 HIS 50 h 0.1000 117CG 47.997627258 39.545143127 67.956726074 HIS 50 c5 0.1000 118ND1 46.669281006 39.956676483 67.916121338 HIS 50 np -0.4200 119CE1 46.730144501 40.789539337 66.829002380 HIS 50 c5 0.2700 120NE2 47.911670685 40.950614929 66.152328491 HIS 50 np -0.5000 121CD2 48.729324341 40.126430511 66.904235840 HIS 50 c5 0.0100 122HE1 45.843067169 41.327060699 66.517631531 HIS 50 h 0.1300 123HE2 48.138290405 41.548683167 65.349815369 HIS 50 hn 0.2800 124HD2 49.789726257 39.981491089 66.738136292 HIS 50 h 0.1300 125N 51.307849884 40.071182251 71.317932129 PRO 51 n -0.4200 126CA 52.692558289 40.596851349 71.184913635 PRO 51 ca 0.0600 127HHA 52.742668152 41.390510559 70.412712097 PRO 51 h 0.1000 128CD 50.980678558 39.703777313 72.706970215 PRO 51 c2 0.0600 129HD1 50.998199463 36.605384827 72.818397522 PRO 51 h 0.1000 130HD2 49.987606049 40.071315765 73.019950867 PRO 51 h 0.1000 131C 53.739063263 39.471630096 70.880722046 PRO 51 c′ 0.3800 132O 53.708900452 38.394466400 71.488830566 PRO 51 o′ -0.3800 133CB 52.868911743 41.240936279 72.572486877 PRO 51 c2 -0.2000 134HB1 53.929355621 41.364253998 72.864852905 PRO 51 h 0.1000 135HB2 52.429229736 42.258647919 72.565872192 PRO 51 h 0.1000 136CG 52.087848663 40.349472046 73.547019958 PRO 51 c2 -0.2000 137HG1 52.750400543 39.566276550 73.963310242 PRO 51 h 0.1000 138HG2 51.686916351 40.923263550 74.403717041 PRO 51 h 0.1000 139N 54.676445007 39.726749420 69.946899414 LEU 52 n -0.5000 140CA 55.768096924 38.764469147 69.570259094 LEU 52 ca 0.1200 141HN 54.573589325 40.637012482 69.488586426 LEU 52 hn 0.2800 142HA 56.414031982 39.325927734 68.869346619 LEU 52 h 0.1000 143C 55.281269073 37.540004730 68.718757629 LEU 52 c′ 0.3800 144O 55.654800415 37.411125183 67.550910950 LEU 52 o′ -0.3800 145CB 56.713882446 38.354763031 70.751991272 LEU 52 c2 -0.2000 146HB1 56.125553131 37.737205505 71.456863403 LEU 52 h 0.1000 147HB2 57.488136292 37.658962250 70.374801636 LEU 52 h 0.1000 148CG 57.411731720 39.487998962 71.552589417 LEU 52 c1 -0.1000 149HG 56.652648926 40.244640350 71.834617615 LEU 52 h 0.1000 150CD1 58.010108948 38.936943054 72.859535217 LEU 52 c3 -0.3000 151HD11 58.475826263 39.735752106 73.467353821 LEU 52 h 0.1000 152HD12 57.236072540 38.469894409 73.497505188 LEU 52 h 0.1000 153HD13 58.787303925 38.171112061 72.675750732 LEU 52 h 0.1000 154CD2 58.517623901 40.187110901 70.742630005 LEU 52 c3 -0.3000 155HD21 58.993679047 41.001243591 71.321037292 LEU 52 h 0.1000 156HD22 59.321178436 39.487018585 70.445312500 LEU 52 h 0.1000 157HD23 58.125030518 40.647811890 69.818283081 LEU 52 h 0.1000 158N 54.475013733 36.643035889 69.315246582 ALA 53 n -0.5000 159CA 53.896503448 35.451416016 68.639259338 ALA 53 ca 0.1200 160HN 54.100524902 37.007503510 70.205230713 ALA 53 hn 0.2800 161HA 53.553531647 35.773445129 67.634338379 ALA 53 h 0.1000 162C 52.602260590 34.908508301 69.353744507 ALA 53 c′ 0.3800 163O 51.589202881 34.828308105 68.650024414 ALA 53 o′ -0.3800 164CB 54.970031738 34.364234924 68.394615173 ALA 53 c3 -0.3000 165HR1 54.534633636 33.449993134 67.949813843 ALA 53 h 0.1000 166HB2 55.742454529 34.720954895 67.688003540 ALA 53 h 0.1000 167HB3 55.500236511 34.068145752 69.316299438 ALA 53 h 0.1000 168N 52.528438568 34.519321442 70.670852661 PRO 54 n -0.4200 169CA 51.288402557 33.944232941 71.268875122 PRO 54 ca 0.0600 170HA 50.860397339 33.194515228 70.573081970 PRO 54 h 0.1000 171CD 53.678298950 34.513732910 71.601814270 PRO 54 c2 0.0600 172HD1 54.146690369 35.509193420 71.717323303 PRO 54 h 0.1000 173HD2 54.456859589 33.804187775 71.264945984 PRO 54 h 0.1000 174C 50.163700104 34.973747253 71.631500244 PRO 54 c′ 0.3800 175O 50.351651196 36.186335425 71.709472656 PRO 54 o′ -0.3500 176CB 51.868888855 33.209735670 72.497810364 PRO 54 c2 -0.2000 177HB1 51.140216827 33.071922302 73.319641113 PRO 54 h 0.1000 178HB2 52.201725006 32.193626404 72.207275391 PRO 54 h 0.1000 179CG 53.074722290 34.047100067 72.925216675 PRO 54 c2 -0.2000 180HG1 52.742275238 34.920612335 73.520271301 PRO 54 h 0.1000 181HG2 53.794158936 33.482940674 73.547462463 PRO 54 h 0.1000 182N 48.950717926 34.451553345 71.882225037 GLY 55 n -0.5000 183CA 47.799011230 35.264900208 72.354187012 GLY 55 cg 0.0200 184HN 48.913242340 33.427757263 71.833122253 GLY 55 hn 0.2800 185HA1 47.829734602 36.291049957 71.943481445 GLY 55 h 0.1000 186HA2 46.873668671 34.838825226 71.925086975 GLY 55 h 0.1000 187C 47.624210358 35.262092590 73.898422241 GLY 55 c′ 0.3800 188O 47.184692383 34.228916168 74.411125183 GLY 55 o′ -0.3800 189N 47.910079956 36.345748901 74.679351807 PRO 56 n -0.4200 190CA 47.789894104 36.319248199 76.162750244 PRO 56 ca 0.0600 191HA 48.177116394 35.361667633 76.567420959 PRO 56 h 0.1000 192CD 48.602729797 37.547939301 74.184982300 PRO 56 c2 0.0600 193HD1 48.046642303 38.065422058 73.380996704 PRO 56 h 0.1000 194HD2 49.595470428 37.261718750 73.794586182 PRO 56 h 0.1000 195C 46.326736450 36.529109955 76.663719177 PRO 56 c′ 0.3800 196O 45.857757568 37.657508850 76.842826843 PRO 56 o′ -0.3800 197CB 48.777050018 37.430667877 76.578651428 PRO 56 c2 -0.2000 198HB1 48.524734497 37.898471832 77.549873352 PRO 56 h 0.1000 199HB2 49.795513153 37.009433746 76.691307068 PRO 56 h 0.1000 200CG 48.752750397 38.431644440 75.422836304 PRO 56 c2 -0.2000 201HG1 47.879917145 39.105598450 75.522354126 PRO 56 h 0.1000 202HG2 49.655212402 39.069591522 75.391311646 PRO 56 h 0.1000 203N 45.616943359 35.415058136 76.897827148 HIS 57 n -0.5000 204HN 46.014194489 34.579135895 76.450401306 HIS 57 hn 0.2800 205CA 44.212440491 35.434158325 77.393867493 HIS 57 ca 0.1200 206HA 43.635601044 36.135776520 76.762809753 HIS 57 h 0.1000 207C 44.146274567 35.882919312 78.904235840 HIS 57 c′ 0.3800 208O 44.718753815 35.174930573 79.742973328 HIS 57 o′ -0.3800 209CB 43.577262878 34.025093079 77.198188782 HIS 57 c2 -0.2000 210HB1 44.242053986 33.250709534 77.626487732 HIS 57 h 0.1000 211HB2 42.665222166 33.964206696 77.822631836 HIS 57 h 0.1000 212CG 43.190551758 33.606594086 75.770996094 HIS 57 c5 0.1000 213HD1 44.009815216 33.724720001 74.654800415 HIS 57 np -0.4200 214CE1 43.220783234 33.103317261 73.724327087 HIS 57 c5 0.2700 215NE2 42.000507355 32.603866577 74.087806702 HIS 57 np -0.5000 216CD2 42.008693695 32.921348572 75.433784485 HIS 57 c5 0.0100 217HE1 43.579235077 32.995296478 72.708923340 HIS 57 h 0.1300 218HE2 41.324546814 32.061363220 73.538429260 HIS 57 hn 0.2800 219HD2 41.238662720 32.638732910 76.138023376 HIS 57 h 0.1300 220N 43.493415833 37.014366150 79.314620972 PRO 58 n -0.4200 221CA 43.576023102 37.521991730 80.713310242 PRD 58 ca 0.0600 222HA 44.640773773 37.575752258 81.019523621 PRO 58 h 0.1000 223CD 42.828823090 37.960189819 78.395561218 PRO 58 c2 0.0600 224HD1 42.088195801 37.471439362 77.735382080 PRO 58 h 0.1000 225HD2 43.573791504 38.467678070 77.749885559 PRO 58 h 0.1000 226C 42.782455444 36.660888672 81.747703552 PRO 58 c′ 0.3800 227O 41.546211243 36.662284851 81.766304016 PRO 58 o′ -0.3800 228CB 43.067096710 38.972381592 80.563407898 PRO 58 c2 -0.2000 229HB1 42.553604126 39.355644226 81.465919495 PRO 58 h 0.1000 230HB2 43.922630310 39.652961731 80.382720947 PRO 58 h 0.1000 231CG 42.156223297 38.958541870 79.333923340 PRO 58 c2 -0.2000 232HG1 41.146640778 38.599040985 79.611282349 PRO 58 h 0.1000 233HG2 42.036239624 39.956802368 78.871780396 PRO 58 h 0.1000 234N 43.511478424 35.934528351 82.617271423 ALA 59 n -0.5000 235HN 44.509765625 35.905010223 82.386589050 ALA 59 hn 0.2800 236CA 42.913761139 34.928112030 83.544029236 ALA 59 c4 0.1200 237HA 42.154701233 34.341350555 82.987930298 ALA 59 h 0.1000 238C 42.181308746 35.481468201 84.821502686 ALA 59 c′ 0.3800 239O 42.459964752 35.112228394 85.965652466 ALA 59 o′ -0.3800 240CB 44.063701630 33.948829651 83.858291626 ALA 59 c3 -0.3000 241HB1 43.708641052 33.104522705 84.478195190 ALA 59 h 0.1000 242HB2 44.502014160 33.503505707 82.944122314 ALA 59 h 0.1000 243HB3 44.882900238 34.433902740 84.422813416 ALA 59 h 0.1000 244N 41.178901672 36.333106995 84.577079773 ALA 60 n -0.5000 245HN 41.112052917 36.562049866 83.573013306 ALA 60 hn 0.2800 246CA 40.189502716 36.803413391 85.578857422 ALA 60 ca 0.1200 247HA 40.008514404 35.981742859 86.302818298 ALA 60 h 0.1000 248C 38.811019897 37.037807465 84.860046387 ALA 60 c′ 0.3800 249O 37.881835938 36.301105499 85.205276489 ALA 60 o′ -0.3800 250CB 40.746566772 37.985549927 86.401313782 ALA 60 c3 -0.3000 251HB1 39.997776031 38.372333527 87.116531372 ALA 60 h 0.1000 252HB2 41.624504089 37.670467377 86.996170044 ALA 60 h 0.1000 253HB3 41.079444885 38.830841064 85.774902344 ALA 60 h 0.1000 254N 38.609931946 37.922218323 83.826889038 PRO 61 n -0.4200 255CA 37.398490906 37.873073578 82.950836182 PRO 61 ca 0.0600 256HA 36.485267639 37.B39759827 83.576164246 PRO 61 h 0.1000 257CD 39.552070618 39.001556396 83.460945129 PRO 61 c2 0.0600 258HD1 40.590957642 38.653537750 83.311111450 PRO 61 h 0.1000 259HD2 39.564563751 39.780746460 84.247795105 PRO 61 h 0.1000 260C 37.256782532 36.700027466 81.909835815 PRO 61 c′ 0.3800 261O 36.243316650 36.657413483 81.209106445 PRO 61 o′ -0.3800 262CB 37.477272034 39.256717682 82.271400452 PRO 61 c2 -0.2000 263HB1 36.964195251 39.298637390 81.290786743 PRO 61 h 0.1000 264HB2 36.981357574 40.016944885 82.906776428 PRO 61 h 0.1000 265CG 38.972518921 39.562160492 82.163795471 PRO 61 c2 -0.2000 266HG1 39.409160614 39.034259796 81.293624878 PRO 61 h 0.1000 267HG2 39.183399200 40.640045166 82.032295227 PRO 61 h 0.1000 268N 38.213741302 35.753643036 81.804443359 SER 62 n -0.5000 269CA 38.144962311 34.600208282 80.863403320 SER 62 ca 0.1200 270HN 39.009967804 35.895751953 82.434890747 SER 62 hn 0.2800 271HA 37.892318726 34.983673096 79.856529236 SER 62 h 0.1000 272C 37.085021973 33.524326324 81.273231506 SER 62 c′ 0.3800 273O 37.382484436 32.625560760 82.070343018 SER 62 o′ -0.3800 274CB 39.569152832 33.994293213 80.760513306 SER 62 c2 -0.1700 275HB1 39.601100922 33.242229462 79.949050903 SER 62 h 0.1000 276HB2 40.317050934 34.759819031 80.475799561 SER 62 h 0.1000 277OG 39.958038330 33.367904663 81.986091614 SER 62 oh -0.3800 278HG 39.157264709 32.928077698 82.316406250 SER 62 ho 0.3500 279N 35.853912354 33.643447876 80.748901367 SER 63 n -0.5000 280CA 34.681579590 32.893772125 81.280097961 SER 63 ca 0.1200 281HN 35.734226227 34.507610321 80.200576782 SER 63 hn 0.2800 282HA 34.978878021 32.281963348 82.158424377 SER 63 h 0.1000 283C 34.028987885 31.963005066 80.214485168 SER 63 c′ 0.3800 284O 33.624385834 32.404701233 79.134857178 SER 63 o′ -0.3800 285CB 33.674541473 33.937458038 81.815757751 SER 63 c2 -0.1700 286HB1 34.172107697 34.614356995 82.538948059 SER 63 h 0.1000 287HB2 33.303077698 34.594402313 81.003784180 SER 63 h 0.1000 288OG 32.576236725 33.301517487 82.471984863 SER 63 oh -0.3800 289HG 32.084590912 32.806625366 81.807708740 SER 63 ho 0.3500 290N 33.852622986 30.677625656 80.556045532 TRP 64 n -0.5000 291CA 33.070865631 29.709415436 79.732810974 TRP 64 ca 0.1200 292HN 34.153343201 30.435497284 81.506057739 TRP 64 hn 0.2800 293HA 33.375720978 29.840501785 78.676765442 TRP 64 h 0.1000 294C 31.526346207 29.958730698 79.816452026 TRP 64 c′ 0.3800 295O 30.936735153 29.911306381 80.900970459 TRP 64 o′ -0.3800 296CB 33.498836517 28.253648758 80.086425781 TRP 64 c2 -0.2000 297HB1 32.936897276 27.550512314 79.442245483 TRP 64 h 0.1000 298HB2 34.548812866 26.110004429 79.767990112 TRP 64 h 0.1000 299CG 33.372638702 27.788063049 81.551361084 TRP 64 c5 0.0000 300C01 32.236022949 27.198472977 62.145393372 TRP 64 c5 0.0100 301NE1 32.442039490 26.926628113 83.513259888 TRP 64 np -0.5000 302CE2 33.734771729 27.365074158 83.750877380 TRP 64 c5 0.1100 303CD2 34.313194275 27.885219574 82.565856934 TRP 64 c5 0.0000 304HD1 31.308589935 27.011001587 81.622375488 TRP 64 h 0.1000 305HE1 31.781488419 26.532098770 84.192108154 TRP 64 hn 0.2800 306CE3 35.633460999 28.410655975 82.581642151 TRP 64 cp -0.1000 307NE3 36.086208344 28.812973022 81.686943054 TRP 64 h 0.1000 308CZ3 36.338203430 28.401332855 83.786201477 TRP 64 cp -0.1000 309HZ3 37.342418671 28.800062180 83.816780090 TRP 64 h 0.1000 310CH2 35.766292572 27.887487411 84.956939697 TRP 64 cp -0.1000 311HH2 36.336753845 27.895225525 85.875114441 TRP 64 h 0.1000 312CZ2 34.469055176 27.367534637 84.959693909 TRP 64 cp -0.1000 313HZ2 34.033626556 26.978828430 85.868453979 TRP 64 h 0.1000 314N 30.884126663 30.253189087 78.672058105 GLY 65 n -0.5000 315CA 29.433704376 30.582933426 78.625785828 GLY 65 cg 0.0200 316HN 31.490442276 30.324731827 77.837860107 GLY 65 hn 0.2800 317HA1 29.049486160 30.927183151 79.604759216 GLY 65 h 0.1000 318HA2 29.301883698 31.462034225 77.967575073 GLY 65 h 0.1000 319C 28.566276550 29.436250687 78.049354553 GLY 65 c′ 0.3800 320O 28.476503372 29.383417130 76.820671082 GLY 65 o′ -0.3800 321N 27.919076920 28.520057678 78.830680847 PRO 66 n -0.4200 322CA 27.254581451 27.307062149 78.266265869 PRO 66 ca 0.0600 323HA 28.007204056 26.749544144 77.674018860 PRO 66 h 0.1000 324CD 27.931732178 28.547025681 80.307373047 PRO 66 c2 0.0600 325HD1 27.674114227 29.536535263 90.727989197 PRO 66 h 0.1000 326HD2 28.930458069 28.264209747 80.693565369 PRO 66 h 0.1000 327C 25.991449356 27.540506363 77.367637634 PRO 66 c′ 0.3800 328O 25.234470367 28.498056412 77.550445557 PRO 66 o′ -0.3800 329CB 26.947065353 26.487516403 79.540168762 PRO 66 c2 -0.2000 330HB1 26.021696091 25.883453369 79.467781067 PRO 66 h 0.1000 331HB2 27.765922546 25.768106461 79.730659485 PRO 66 h 0.1000 332CG 26.879997253 27.505201340 80.680580139 PRO 66 c2 -0.2000 333HG1 25.878761292 27.974979401 80.712356567 PRO 66 h 0.1000 334HG2 27.057765961 27.053478241 81.674545288 PRO 66 h 0.1000 335N 25.764133453 26.624628067 76.406608582 CYS 67 n -0.5000 336CA 24.578670502 26.665664673 75.512832642 CYS 67 ca 0.1200 337HN 26.474828720 25.893449783 76.320098877 CYS 67 hn 0.2800 338HA 24.437746048 27.701400757 75.152099609 CYS 67 h 0.1000 339C 23.256376266 26.130277634 76.174392700 CYS 67 c′ 0.3800 340O 23.219629288 24.940492630 76.516906738 CYS 67 o′ -0.3800 341CB 24.900175095 25.815908432 74.260444641 CYS 67 c2 -0.3000 342HB1 25.807971954 26.178848267 73.749794006 CYS 67 h 0.1000 343HR2 25.105169296 24.761932373 74.532623291 CYS 67 h 0.1000 344SG 23.472158432 25.844451904 73.133270264 CYS 67 s1 0.1000 345N 22.124137878 26.895683289 76.264381409 PRO 68 n -0.4200 346CA 20.786550522 26.297697067 76.529830933 PRO 68 ca 0.0600 347HA 20.877141953 25.506265640 77.300582886 PRO 68 h 0.1000 348CD 22.161409378 28.364057541 76.432929993 PRO 68 c2 0.0600 349HD1 22.628620148 28.901371002 75.585960388 PRO 68 h 0.1000 350GD2 22.732339859 28.631174088 77.345329285 PRO 68 h 0.1000 351C 20.190311432 25.593938828 75.255737305 PRO 68 c′ 0.3800 352O 20.566764832 24.451507568 74.984413147 PRO 68 o′ -0.3800 353CB 20.033475876 27.483673096 77.173645020 PRO 68 c2 -0.2000 354HB1 18.940057755 27.449979782 77.017181396 PRO 68 h 0.1000 355HB2 20.183664322 27.458950043 78.271354675 PRO 68 h 0.1000 356CG 20.687761307 28.746078491 76.604209900 PRO 68 c2 -0.2000 357HG1 20.234010696 29.017192841 75.632743835 PRO 68 h 0.1000 358HG2 20.559530256 29.623554230 77.265640259 PRO 68 h 0.1000 359H 19.297069550 26.226366043 74.460205078 ARG+ 69 n -0.5000 360CA 18.727945328 25.594141006 73.229873657 ARG+ 69 ca 0.1200 361HN 18.899488449 27.074655533 74.874603271 ARG+ 69 hn 0.2800 362HA 19.468439102 24.889890671 72.798027039 ARG+ 69 h 0.1000 363C 18.426181793 26.666866302 72.127845764 ARG+ 69 c′ 0.3800 364O 17.302417755 27.170328140 72.057907104 ARG+ 69 o′ -0.3800 365CB 17.487716675 24.741283417 73.645401001 ARG+ 69 c2 -0.2000 366HB1 17.790594101 24.038330078 74.447227478 ARG+ 69 h 0.1100 367HB2 16.742151260 25.409061432 74.119949341 ARG+ 69 h 0.1100 368CG 16.806570053 23.930654526 72.510940552 ARG+ 69 c2 -0.2000 369HG1 16.500089645 24.624885559 71.702163696 ARG+ 69 h 0.1300 370HG2 17.539186478 23.235509872 72.053100586 ARG+ 69 h 0.1300 371CD 15.574314117 23.148860931 73.007453918 ARG+ 69 c2 -0.0900 372HD1 15.890284538 22.374292374 73.738624573 ARG+ 69 h 0.1300 373HD2 14.902976036 23.843069077 73.554016113 ARG+ 69 h 0.1300 374NE 14.865127563 22.521680832 71.855873108 ARG+ 69 n1 -0.5000 375HE 15.293711662 22.507183075 70.926025391 ARG+ 69 hn 0.3600 376CZ 13.645489693 21.980854034 71.902374268 ARG+ 69 cr 0.4500 377NH1 13.127552986 21.522832870 70.798370361 ARG+ 69 n2 -0.5000 378H11 13.689088821 21.608518600 69.948852539 ARG+ 69 hn 0.3600 379H12 12.188611031 21.122539520 70.853851318 ARG+ 69 hn 0.3600 380NH2 12.936479568 21.886768341 73.000465393 ARG+ 69 n2 -0.5000 381HH21 12.008401871 21.462900162 72.952354431 ARG+ 69 hn 0.3600 382HH22 13.405644417 22.251142502 73.831558228 ARG+ 69 hn 0.3600 383N 19.430337906 26.966600418 71.273384094 ARG+ 70 n -0.5000 384CA 19.316965103 27.784936905 70.016807556 ARG+ 70 ca 0.1200 385HN 20.317523956 26.522159576 71.527793884 ARG+ 70 hn 0.2800 386HA 19.139877319 27.013025284 69.241798401 ARG+ 70 h 0.1000 387C 20.690151215 28.397680283 69.573341370 ARG+ 70 c′ 0.3800 388O 21.267679214 27.963806152 68.579154968 ARG+ 70 o′ -0.3800 389CB 18.103752136 28.753881454 69.796676636 ARG+ 70 c2 -0.2000 390HB1 18.134477615 29.133897781 68.754875183 ARG+ 70 h 0.1100 391HB2 17.183977127 28.135446548 69.816154480 ARG+ 70 h 0.1100 392CG 17.944366455 29.952959061 70.767364502 ARG+ 70 c2 -0.2000 393HG1 18.201717377 29.630409241 71.795295715 ARG+ 70 h 0.1300 394HG2 18.686578751 30.737569809 70.523498535 ARG+ 70 h 0.1300 395CD 16.516407013 30.528032303 70.757499695 ARG+ 70 c2 -0.0900 396HD1 16.205293655 30.812314987 69.732498169 ARG+ 70 h 0.1300 397HD2 15.804359436 29.724859238 71.041206360 ARG+ 70 h 0.1300 398NE 16.396289825 31.632083893 71.751823425 ARG+ 70 n1 -0.5000 399HE 16.381929398 31.418577194 72.754409790 ARG+ 70 hn 0.3600 400CZ 16.270032883 32.931114197 71.475357056 ARG+ 70 cr 0.4500 401NH1 16.119342804 33.758121490 72.470024109 ARG+ 70 n2 -0.5000 402HH11 16.097789764 33.352241516 73.407394409 ARG+ 70 hn 0.3600 403HH12 16.020824432 34.751869202 72.242614746 ARG+ 70 hn 0.3600 404NH2 16.295974731 33.427260426 70.262794495 ARG+ 70 n2 -0.5000 405HH21 16.191591263 34.436088562 70.142570496 ARG+ 70 hn 0.3600 406HH22 16.439620972 32.732715607 69.527351379 ARG+ 70 hn 0.3600 407N 21.215826035 29.506198883 70.104598999 TYRC 71 n -0.5000 408HN 21.692993164 29.961484909 69.319816589 TYRC 71 hn 0.2800 409CA 22.037544250 29.469150543 71.348724365 TYRC 71 ca 0.1200 410HA 22.727062225 28.601654053 71.296867371 TYRC 71 h 0.1000 411C 21.230804443 29.295030594 72.663772583 TYRC 71 c′ 0.4100 412OXT 20.420524597 30.105148315 73.113685608 TYRC 71 o′ -0.3800 413O 21.522335052 28.107995987 73.273979187 TYRC 71 oh -0.3800 414HO 20.994127274 28.000011444 74.066841125 TYRC 71 ho 0.3500 415CB 22.938613892 30.740638733 71.402084351 TYRC 71 c2 -0.2000 416HB1 22.321226120 31.652799606 71.283157349 TYRC 71 h 0.1000 417HB2 23.363500595 30.853305817 72.420455933 TYRC 71 h 0.1000 418CG 24.110603333 30.760416031 70.402580261 TYRC 71 cp 0.0000 419CD1 23.933057785 31.274461746 69.111869812 TYRC 71 cp -0.1000 420HD1 22.977622986 31.679159164 68.809974670 TYRC 71 h 0.1000 421CE1 24.985538483 31.264329910 68.201301575 TYRC 71 cp -0.1000 422HE1 24.833002090 31.650396347 67.203536987 TYRC 71 h 0.1000 423CZ 26.227394104 30.757091522 68.577186584 TYRC 71 cp 0.0300 424OH 27.265848160 30.762763977 67.686424255 TYRC 71 oh -0.3800 425HH 26.966634750 31.154380798 66.863937378 TYRC 71 ho 0.3500 426CE2 26.415199280 30.251981735 69.859985352 TYRC 71 cp -0.1000 427HE2 27.377700806 29.852491379 70.147377014 TYRC 71 h 0.1000 428CD2 25.360828400 30.253871916 70.770927429 TYRC 71 cp -0.1000 429HD2 25.521846771 29.846044540 71.760574341 TYRC 71 h 0.1000 430
Claims (14)
1.一种可溶性多肽组分，它具有LAG-3蛋白的4种免疫球蛋白类型胞外结构域中的第一种(序列SEQ ID No.1的氨基酸1-159)，其中SEQ ID No.1的73、75和76位置上的一个或多个精氨酸(Arg)残基被谷氨酸(Glu)取代。
2.根据权利要求1的可溶性多肽组分，它另外包含LAG-3蛋白的其它3种免疫球蛋白类型胞外结构域(序列SEQ ID No.1的氨基酸160-239，240-330和331-412)中的一个或多个。
3.根据权利要求1或2的可溶性多肽组分，它进一步包含在其C-末端和/或N-末端的辅助肽序列，以便组成一种融合蛋白。
4.根据权利要求3的可溶性多肽组分，其中所述辅助肽序列包含一种免疫球蛋白的一部分。
5.根据权利要求4的可溶性多肽组分，其中所述辅助肽序列包含一种IgG4同种型的免疫球蛋白的一部分。
6.根据权利要求1-5任一项的可溶性多肽组分，其特征在于，它还同一种毒素或一种放射性同位素结合。
7.生产权利要求4或5的可溶性多肽组分的方法，其特征在于，将编码与LAG-3相应的多肽区的cDNA片段，经PCR扩增之后，和编码免疫球蛋白的相关区的cDNA，其中该cDNA同编码LAG-3的相应多肽区的cDNA融合，被插入一个载体，其特征还在于，转染之后，该cDNA片段在一个表达系统中被表达。
8.根据权利要求7的方法，其中所述表达系统是哺乳动物细胞。
9.根据权利要求8的方法，其中所述细胞是仓鼠卵巢细胞。
10.根据权利要求7-9任一项的生产可溶性肽组分的方法，其特征在于，对构建的LAG-3/免疫球蛋白缀合物进行裂解，以包含一个合适的裂解位点。
11.具有免疫抑制剂活性的治疗组合物，它包含权利要求1-6任一项的可溶性多肽组分。
12.特异性地抗权利要求1-6任一项的可溶性多肽组分的多克隆或单克隆抗体。
13.具有免疫刺激活性的治疗组合物，它包含一种特异性地抗权利要求1-6任一项的可溶性多肽组分的抗体。
14.权利要求13的治疗组合物，其中所述抗体进一步与一种细胞毒素或一种放射性同位素结合。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR94/05643 | 1994-05-06 | ||
| FR9405643 | 1994-05-06 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1155904A CN1155904A (zh) | 1997-07-30 |
| CN1110557C true CN1110557C (zh) | 2003-06-04 |
Family
ID=9463004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95193718A Expired - Lifetime CN1110557C (zh) | 1994-05-06 | 1995-05-05 | Lag－3蛋白的可溶性多肽组分;生产方法、治疗组合物、抗独特型抗体 |
Country Status (18)
| Country | Link |
|---|---|
| US (3) | US5955300A (zh) |
| EP (1) | EP0758383B1 (zh) |
| JP (1) | JP3700859B2 (zh) |
| KR (1) | KR100257466B1 (zh) |
| CN (1) | CN1110557C (zh) |
| AT (1) | ATE352617T1 (zh) |
| BR (1) | BR9507618A (zh) |
| CA (1) | CA2189657C (zh) |
| DE (1) | DE69535375T2 (zh) |
| DK (1) | DK0758383T3 (zh) |
| ES (1) | ES2281899T3 (zh) |
| IL (1) | IL113617A (zh) |
| MX (1) | MX9605365A (zh) |
| NO (1) | NO325828B1 (zh) |
| PT (1) | PT758383E (zh) |
| RU (1) | RU2178306C2 (zh) |
| WO (1) | WO1995030750A2 (zh) |
| ZA (1) | ZA953629B (zh) |
Families Citing this family (48)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2227334A1 (en) * | 1995-07-21 | 1997-02-06 | Applied Research Systems Ars Holding N.V. | Methods for detecting, identifying, isolating, and selectively labelling and targeting th1 lymphocytes by means of the lag-3 protein |
| WO1998023741A1 (fr) * | 1996-11-28 | 1998-06-04 | Institut Gustave Roussy | Mutants de la proteine lag-3, leur expression et utilisation |
| EP0941329B1 (en) | 1996-11-29 | 2004-07-21 | Applied Research Systems ARS Holding N.V. | Methods for preventing graft rejection in transplantation and for producing a universal gene therapy host cell using lymphocyte activation (lag-3) |
| EP0900841A1 (en) * | 1997-06-18 | 1999-03-10 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | LAG-3 splice variants |
| EP0893507A1 (en) | 1997-07-25 | 1999-01-27 | Institut Gustave Roussy | Use of MHC class II ligands (CD4 and LAG-3) as adjuvant for vaccination and of LAG-3 in cancer treatment |
| US6660843B1 (en) † | 1998-10-23 | 2003-12-09 | Amgen Inc. | Modified peptides as therapeutic agents |
| US6434012B2 (en) * | 2000-02-11 | 2002-08-13 | Tyco Electronics Logistics Ag | Circuit board interconnect |
| AU2001259432B2 (en) * | 2000-05-03 | 2005-04-21 | Amgen Inc. | Modified peptides, comprising an FC domain, as therapeutic agents |
| JP2005535290A (ja) * | 2002-02-22 | 2005-11-24 | ジェネンテック・インコーポレーテッド | 免疫関連疾患の治療のための組成物と方法 |
| EP1596871A4 (en) | 2003-02-28 | 2006-08-23 | Drew M Pardoll | REGULATION BY LYMPHOCYTES T |
| FR2868781B1 (fr) * | 2004-04-13 | 2008-02-22 | Immutep | Composition de vaccin comprenant un ligand cmh de classe ii couple a un antigene, procede de preparation et utilisations |
| EP2044949A1 (en) | 2007-10-05 | 2009-04-08 | Immutep | Use of recombinant lag-3 or the derivatives thereof for eliciting monocyte immune response |
| AR072999A1 (es) | 2008-08-11 | 2010-10-06 | Medarex Inc | Anticuerpos humanos que se unen al gen 3 de activacion linfocitaria (lag-3) y los usos de estos |
| UY34887A (es) | 2012-07-02 | 2013-12-31 | Bristol Myers Squibb Company Una Corporacion Del Estado De Delaware | Optimización de anticuerpos que se fijan al gen de activación de linfocitos 3 (lag-3) y sus usos |
| ES2728578T3 (es) | 2013-09-20 | 2019-10-25 | Bristol Myers Squibb Co | Combinación de anticuerpos anti-LAG-3 y anticuerpos anti-PD-1 para tratar tumores |
| GB201322626D0 (en) | 2013-12-19 | 2014-02-05 | Immutep S A | Combined preparations for the treatment of cancer |
| JOP20200094A1 (ar) | 2014-01-24 | 2017-06-16 | Dana Farber Cancer Inst Inc | جزيئات جسم مضاد لـ pd-1 واستخداماتها |
| PT3116909T (pt) | 2014-03-14 | 2020-01-30 | Novartis Ag | Moléculas de anticorpos para lag-3 e suas utilizações |
| NZ726513A (en) | 2014-05-28 | 2023-07-28 | Memorial Sloan Kettering Cancer Center | Anti-gitr antibodies and methods of use thereof |
| TWI693232B (zh) | 2014-06-26 | 2020-05-11 | 美商宏觀基因股份有限公司 | 與pd-1和lag-3具有免疫反應性的共價結合的雙抗體和其使用方法 |
| JO3663B1 (ar) | 2014-08-19 | 2020-08-27 | Merck Sharp & Dohme | الأجسام المضادة لمضاد lag3 وأجزاء ربط الأنتيجين |
| GB201500374D0 (en) | 2015-01-09 | 2015-02-25 | Immutep S A | Combined preparations for the treatment of cancer |
| MA41463A (fr) | 2015-02-03 | 2017-12-12 | Anaptysbio Inc | Anticorps dirigés contre le gène d'activation 3 des lymphocytes (lag-3) |
| TWI773646B (zh) | 2015-06-08 | 2022-08-11 | 美商宏觀基因股份有限公司 | 結合lag-3的分子和其使用方法 |
| CN115887671A (zh) | 2015-07-16 | 2023-04-04 | 百欧肯治疗有限公司 | 治疗癌症的组合物及方法 |
| EP3328419B1 (en) | 2015-07-30 | 2021-08-25 | MacroGenics, Inc. | Pd-1-binding molecules and methods of use thereof |
| WO2017025871A1 (en) | 2015-08-07 | 2017-02-16 | Glaxosmithkline Intellectual Property Development Limited | Combination therapy comprising anti ctla-4 antibodies |
| TWI756187B (zh) | 2015-10-09 | 2022-03-01 | 美商再生元醫藥公司 | 抗ｌａｇ３抗體及其用途 |
| RS61866B1 (sr) * | 2015-11-20 | 2021-06-30 | Regeneron Pharma | Ne-humane životinje koje imaju humanizovani gen za aktivaciju limfocita 3 |
| AU2016364889B2 (en) | 2015-12-02 | 2024-01-04 | Agenus Inc. | Antibodies and methods of use thereof |
| AU2016370376B2 (en) | 2015-12-14 | 2023-12-14 | Macrogenics, Inc. | Bispecific molecules having immunoreactivity with PD-1 and CTLA-4, and methods of use thereof |
| AU2016371639A1 (en) | 2015-12-16 | 2018-06-28 | Merck Sharp & Dohme Llc | Anti-LAG3 antibodies and antigen-binding fragments |
| CA3012833A1 (en) * | 2016-02-04 | 2017-08-10 | Trianni, Inc. | Enhanced production of immunoglobulins |
| MX2018016364A (es) | 2016-06-20 | 2019-11-28 | Kymab Ltd | Anticuerpos anti-pd-l1. |
| JP6998895B2 (ja) | 2016-06-23 | 2022-01-18 | 江蘇恒瑞医薬股▲ふん▼有限公司 | Ｌａｇ－３抗体、その抗原結合フラグメント、およびそれらの医薬的用途 |
| WO2018071500A1 (en) | 2016-10-11 | 2018-04-19 | Agenus Inc. | Anti-lag-3 antibodies and methods of use thereof |
| WO2018069500A2 (en) | 2016-10-13 | 2018-04-19 | Symphogen A/S | Anti-lag-3 antibodies and compositions |
| UY37463A (es) | 2016-11-02 | 2018-05-31 | Glaxosmithkline Ip No 2 Ltd | Proteínas de unión |
| EP3580238A1 (en) | 2017-02-10 | 2019-12-18 | Regeneron Pharmaceuticals, Inc. | Radiolabeled anti-lag3 antibodies for immuno-pet imaging |
| MA49042A (fr) | 2017-04-05 | 2020-02-12 | Symphogen As | Polythérapies ciblant pd-1, tim-3 et lag-3 |
| WO2018208868A1 (en) * | 2017-05-10 | 2018-11-15 | Smet Pharmaceutical Inc | Human monoclonal antibodies against lag3 and uses thereof |
| CA3060984A1 (en) | 2017-05-30 | 2018-12-06 | Bristol-Myers Squibb Company | Treatment of lag-3 positive tumors |
| EP3630179A2 (en) | 2017-05-30 | 2020-04-08 | Bristol-Myers Squibb Company | Compositions comprising an anti-lag-3 antibody or an anti-lag-3 antibody and an anti-pd-1 or anti-pd-l1 antibody |
| WO2019046225A1 (en) | 2017-08-30 | 2019-03-07 | Phanes Therapeutics, Inc. | ANTI-LAG-3 ANTIBODIES AND USES THEREOF |
| KR20200104314A (ko) | 2017-12-22 | 2020-09-03 | 지앙수 헨그루이 메디슨 컴퍼니 리미티드 | Lag-3 항체 약학 조성물 및 이의 용도 |
| MX2021013815A (es) | 2019-05-13 | 2021-12-14 | Regeneron Pharma | Combinacion de inhibidores de pd-1 e inhibidores de lag-3 para mejorar la eficacia en el tratamiento contra el cancer. |
| CN112010972B (zh) * | 2019-05-31 | 2023-01-10 | 瑞阳(苏州)生物科技有限公司 | 与人lag-3蛋白结合的抗体及其编码基因和应用 |
| CN110950966B (zh) * | 2019-12-13 | 2020-12-11 | 启辰生生物科技(珠海)有限公司 | 融合蛋白、编码核酸和细胞及用途 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992000092A1 (en) * | 1990-07-02 | 1992-01-09 | Bristol-Myers Squibb Company | Ligand for cd28 receptor on b cells and methods |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE721983T1 (de) | 1988-01-22 | 2002-07-04 | Zymogenetics Inc | Verfahren zur herstellung von biologisch-aktive Dimerpeptiden |
| US5116964A (en) | 1989-02-23 | 1992-05-26 | Genentech, Inc. | Hybrid immunoglobulins |
| US5976877A (en) | 1990-01-08 | 1999-11-02 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Proteins produced by human lymphocytes DNA sequence encoding these proteins and their pharmaceutical and biological uses |
| FR2656800B1 (fr) | 1990-01-08 | 1992-05-15 | Roussy Inst Gustave | Nouvelles proteines produits par les lymphocytes humains, sequence d'adn codant pour ces proteines et applications pharmaceutiques et biologiques. |
-
1995
- 1995-05-05 KR KR1019960706281A patent/KR100257466B1/ko not_active IP Right Cessation
- 1995-05-05 DK DK95920125T patent/DK0758383T3/da active
- 1995-05-05 US US08/737,271 patent/US5955300A/en not_active Ceased
- 1995-05-05 MX MX9605365A patent/MX9605365A/es active IP Right Grant
- 1995-05-05 ZA ZA953629A patent/ZA953629B/xx unknown
- 1995-05-05 EP EP95920125A patent/EP0758383B1/fr not_active Expired - Lifetime
- 1995-05-05 ES ES95920125T patent/ES2281899T3/es not_active Expired - Lifetime
- 1995-05-05 BR BR9507618A patent/BR9507618A/pt not_active IP Right Cessation
- 1995-05-05 AT AT95920125T patent/ATE352617T1/de active
- 1995-05-05 IL IL113617A patent/IL113617A/en not_active IP Right Cessation
- 1995-05-05 DE DE69535375T patent/DE69535375T2/de not_active Expired - Lifetime
- 1995-05-05 WO PCT/FR1995/000593 patent/WO1995030750A2/fr active IP Right Grant
- 1995-05-05 PT PT95920125T patent/PT758383E/pt unknown
- 1995-05-05 JP JP52872995A patent/JP3700859B2/ja not_active Expired - Lifetime
- 1995-05-05 CN CN95193718A patent/CN1110557C/zh not_active Expired - Lifetime
- 1995-05-05 RU RU96123299/14A patent/RU2178306C2/ru active
- 1995-05-05 CA CA002189657A patent/CA2189657C/fr not_active Expired - Lifetime
- 1995-05-05 US US09/931,103 patent/USRE38313E1/en not_active Expired - Lifetime
-
1996
- 1996-11-04 NO NO19964650A patent/NO325828B1/no not_active IP Right Cessation
-
1998
- 1998-04-13 US US09/058,555 patent/US6143273A/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992000092A1 (en) * | 1990-07-02 | 1992-01-09 | Bristol-Myers Squibb Company | Ligand for cd28 receptor on b cells and methods |
Also Published As
| Publication number | Publication date |
|---|---|
| PT758383E (pt) | 2007-05-31 |
| CN1155904A (zh) | 1997-07-30 |
| EP0758383A1 (fr) | 1997-02-19 |
| IL113617A (en) | 2007-09-20 |
| DE69535375D1 (de) | 2007-03-15 |
| BR9507618A (pt) | 1997-08-19 |
| AU2570195A (en) | 1995-11-29 |
| ATE352617T1 (de) | 2007-02-15 |
| ES2281899T3 (es) | 2007-10-01 |
| NO325828B1 (no) | 2008-07-28 |
| RU2178306C2 (ru) | 2002-01-20 |
| DE69535375T2 (de) | 2007-11-08 |
| EP0758383B1 (fr) | 2007-01-24 |
| NO964650D0 (no) | 1996-11-04 |
| KR100257466B1 (ko) | 2000-07-01 |
| CA2189657C (fr) | 2002-03-12 |
| NO964650L (no) | 1997-01-06 |
| ZA953629B (en) | 1996-11-05 |
| CA2189657A1 (fr) | 1995-11-16 |
| AU708825B2 (en) | 1999-08-12 |
| WO1995030750A3 (fr) | 1995-12-21 |
| US6143273A (en) | 2000-11-07 |
| IL113617A0 (en) | 1995-08-31 |
| DK0758383T3 (da) | 2007-05-29 |
| WO1995030750A2 (fr) | 1995-11-16 |
| WO1995030750A8 (fr) | 1999-07-29 |
| MX9605365A (es) | 1997-12-31 |
| JPH09508023A (ja) | 1997-08-19 |
| USRE38313E1 (en) | 2003-11-11 |
| US5955300A (en) | 1999-09-21 |
| JP3700859B2 (ja) | 2005-09-28 |
| KR970702917A (ko) | 1997-06-10 |
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