CN108101988A - 针对cd16的全人源单域抗体、其抗原结合片段及应用 - Google Patents
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Abstract
本发明属于生物技术领域，具体涉及针对CD16的全人源单域抗体、其抗原结合片段，以及它们在制备用于疾病诊断和治疗制剂中的应用。本发明公开了能特异性结合CD16的全人源单域抗体及其抗原结合片段。其主要特征在于此单域抗体是由存在于抗体重链基因可变区中的互补决定区（complementarity‑determining region，CDR）特异性基因序列决定的，并在原核和真核细胞中获得有效表达的特异性结合CD16的抗体。利用此抗体CDR区或部分或全基因，可在原核和真核细胞及任何表达系统中改造和生产不同形式的基因工程抗体，可用于制备在临床上诊断或治疗相关疾病的制剂。
Description
技术领域
本发明属于生物技术领域，具体涉及针对CD16的全人源单域抗体、其抗原结合片段，以及它们在制备用于疾病诊断和治疗制剂中的应用。
背景技术
现有技术公开了CD16（也称作FcRγIII）是IgGs的Fc片段具有低亲和力的受体，该受体能够引发自然杀伤细胞（natural killer cells，NKs）细胞对靶细胞的溶解。有研究表明，人NK细胞大约包含15%的淋巴细胞，大多数的NK细胞CD56的表达量低（CD56dim），而CD16表达水平高。人CD16具有CD16A和CD16B两种亚型，两者在结构上表现了胞外免疫球蛋白结合区域序列具有96%的序列同一性。CD16A是巨噬细胞，肥大细胞以及NK细胞表达的跨膜受体，与CD3ζ和Fc-εRI-γ共定位于NK细胞上，一旦CD16A的α链与含有Fc-εRI-γ链的免疫受体酪氨酸激活基序（immunoreceptor tyrosine-based activation motif，ITAM）和/或T细胞受体（T cell receptor，TCR）/CD3ζ结合，诱发信号传导，导致细胞因子的产生和细胞毒性作用。CA16B作为糖基-磷脂酰肌醇（glycosylphosphatidylinositol，GPI）锚定受体存在于多核粒细胞上，该受体不能引发效应细胞对肿瘤细胞的杀伤作用；此外，所述CD16B以可溶性受体存在于血清中，一旦与抗体结合形成免疫复合物会引起副作用。
有研究表明，表达CD16的免疫效应细胞与抗体的Fc段识别结合后，使自身活化，诱导抗体依赖细胞毒性（antibody-dependent cellular cytotoxicity，ADCC）从而杀伤肿瘤细胞，其中NK细胞是介导ADCC效应的最主要免疫效应细胞；然而，在大多数肿瘤中包括肠癌，肾癌，肝癌，乳腺癌和肺癌发现其肿瘤微环境中NK细胞的数目极少。因此，业内发展一种利用CD16的活性来治疗癌症的免疫治疗方法，如具有肿瘤表面抗原特异性的抗体以及NK细胞CD16特异性的抗体形成的双特异性分子，目前已经开发了HER2/neu和CD16的双特异性抗体，比单独的HER2抗体具有更强的抗肿瘤活性；HRS-3/A9，一种特异性结合CD16和CD30的双特异性抗体，通过结合NK细胞或单核细胞表面的CD16分子，使这些免疫效应细胞靶向表达CD30的肿瘤细胞进行杀伤，该抗体不论在霍奇金氏瘤的小鼠模型中还是患者中都可完全清除肿瘤，，然而，这些鼠源性抗体在人体内半衰期短。
因此，研发高效、特异性的抗CD16的全人源单域抗体意义重大；这些单域抗体不仅可用于的体外的快速检测，也可以用于自身免疫疾病和癌症的治疗。
与本发明相关的现有技术有，
[1] Yeap WH, Wong KL, Shimasaki N, et al. CD16 is indispensable forantibody-dependent cellular cytotoxicity by human monocytes. Sci Rep. 2016;6:34310.
[2] van de Winkel JG, Capel PJ. Human IgG Fc receptor heterogeneity:molecular aspects and clinical implications. Immunol Today. 1993;14(5):215-21.
[3] Perussia B. Fc receptors on natural killer cells. Curr Top MicrobiolImmunol. 1998, 230: 63-88.
[4] Kudo K, Imai C, Lorenzini P, et al. T lymphocytes expressing a CD16signaling receptor exert antibody-dependent cancer cell killing. Cancer Res.2014;74(1):93-103.
[5] Shahied LS, Tang Y, Alpaugh RK, et al. Bispecific minibodiestargeting HER2/neu and CD16 exhibit improved tumor lysis when placed in adivalent tumor antigen binding format. J Biol Chem. 2004;279(52):53907-14.
[6] Hombach A, Jung W, Pohl C, et al. A CD16/CD30 bispecific antibodyinduces lysis of Hodgkin cells by unstimulated natural killer cells in vitroand in vivo. Int J Cancer. 1993;55:830-6.
[7] Hartmann F, Renner C, Jung W, et al. Anti-CD16/CD30 bispecificantibody treatment for Hodgkin's disease: role of infusion schedule andcostimulation with cytokines. Clin Cancer Res. 2001;7(7):1873-81.
[8] Ying T, Du L, Ju TW, et al. Exceptionally potent neutralization ofMiddle East respiratory syndrome coronavirus by human monoclonal antibodies.J Virol. 2014;88(14):7796-805.。
发明内容
本发明的目的是基于现有技术的现状，提供针对CD16的全人源单域抗体、其抗原结合片段及应用。
本发明提供了特异性结合CD16的全人源单域抗体；所述的的全人源单域抗体包括n111，n112，n113，n114，n115，n116，n117和n118；本发明还公开了所述抗体的抗原结合片段，双特异性抗体，与效应分子的免疫偶联物，及融合蛋白及组合物；所述的抗体和组合物可以用于制备用于疾病的诊断和治疗的制剂。
在本发明的一些实施方式中，单域抗体重链可变区的氨基酸序列，包含至少一个（比如三个）n111，n112，n113，n114，n115，n116，n117和n118的重链可变区的CDR区；其中，
SEQ ID NO: 1 是n111重链可变区的氨基酸序列；
SEQ ID NO: 2 是n111重链可变区CDR1的氨基酸序列；
SEQ ID NO: 3 是n111重链可变区CDR2的氨基酸序列；
SEQ ID NO: 4 是n111重链可变区CDR3的氨基酸序列；
SEQ ID NO: 5 是n112重链可变区的氨基酸序列；
SEQ ID NO: 6 是n112重链可变区CDR1的氨基酸序列；
SEQ ID NO: 7 是n112重链可变区CDR2的氨基酸序列；
SEQ ID NO: 8 是n112重链可变区CDR3的氨基酸序列。
SEQ ID NO: 9 是n113重链可变区的氨基酸序列；
SEQ ID NO: 10 是n113重链可变区CDR1的氨基酸序列；
SEQ ID NO: 11 是n113重链可变区CDR2的氨基酸序列；
SEQ ID NO: 12 是n113重链可变区CDR3的氨基酸序列。
SEQ ID NO: 13 是n114重链可变区的氨基酸序列；
SEQ ID NO: 14 是n114重链可变区CDR1的氨基酸序列；
SEQ ID NO: 15 是n114重链可变区CDR2的氨基酸序列；
SEQ ID NO: 16 是n114重链可变区CDR3的氨基酸序列；
SEQ ID NO: 17 是n115重链可变区的氨基酸序列;
SEQ ID NO: 18 是n115重链可变区CDR2的氨基酸序列；
SEQ ID NO: 19 是n115重链可变区CDR1的氨基酸序列；
SEQ ID NO: 20 是n115重链可变区CDR3的氨基酸序列。
SEQ ID NO: 21 是n116重链可变区的氨基酸序列；
SEQ ID NO: 22 是n116重链可变区CDR1的氨基酸序列；
SEQ ID NO: 23 是n116重链可变区CDR2的氨基酸序列；
SEQ ID NO: 24 是n116重链可变区CDR3的氨基酸序列；
SEQ ID NO: 25 是n118重链可变区的氨基酸序列；
SEQ ID NO: 26 是n118重链可变区CDR1的氨基酸序列；
SEQ ID NO: 27 是n118重链可变区CDR2的氨基酸序列；
SEQ ID NO: 28 是n118重链可变区CDR3的氨基酸序列。
SEQ ID NO: 29 是n117重链可变区的氨基酸序列；
SEQ ID NO: 30 是n117重链可变区CDR1的氨基酸序列；
SEQ ID NO: 31 是n117重链可变区CDR2的氨基酸序列；
SEQ ID NO: 32 是n117重链可变区CDR3的氨基酸序列。
本发明还公开了编码这些单域抗体和抗原结合片段的核酸；在本发明一些实施方案中，公开了包含这些核酸的载体，及包含这些载体的宿主细胞；在进一步的实施方案中，公开了包含这些单域抗体，抗原结合片段，双特异性抗体，免疫偶联物，融合蛋白，核酸，质粒，和载体的药用组合物；在其它实施方案中，这些单域抗体，抗原结合片段，双特异性抗体，免疫偶联物，融合蛋白，核酸，质粒，和载体被用来制备诊断或治疗自身免疫疾病或癌症等相关疾病的制剂。
本发明中，所述的针对CD16蛋白的全人源单域抗体，或抗原结合片段，包含：
抗体的重链可变区含有SEQ ID NO:1的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:5的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:9的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:13的26-33位，51-58位，和/或97-105位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:17的26-33位，51-58位，和/或97-108位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:21的26-33位，51-57位，和/或96-110位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:25的26-33位，51-57位，和/或96-111位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:29的26-33位，51-57位，和/或96-102位氨基酸残基，
其中单域抗体或抗原结合片段特异性结合CD16。
优选的，本发明所述的单域抗体或抗原结合片段，包含：
抗体的重链可变区含有SEQ ID NO:1的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:5的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:9的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:13的26-33位，51-58位，和/或97-105位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:17的26-33位，51-58位，和/或97-108位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:21的26-33位，51-57位，和/或96-110位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:25的26-33位，51-57位，和/或96-111位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:29的26-33位，51-57位，和/或96-102位氨基酸残基。
本发明中，所述的单域抗体或抗原结合片段，其中：
重链可变区的氨基酸序列与SEQ ID NO:1具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:5具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:9具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:13具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:17具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:21具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:25具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:29具有至少90%的相同性。
本发明中，所述的单域抗体或抗原结合片段是全人源的；其中的单域抗体为VH，抗原结合片段为VH。
本发明还提供了含有上述的单域抗体或抗原结合片段的双特异性抗体。
本发明中，所述的单域抗体或抗原结合片段或所述的双特异性抗体，与一种效应分子偶联，其中所述的效应分子是可检测标记，优选效应分子是毒素或化疗剂；所述的可检测标记是荧光标记，放射性标记，亲和素，生物素，或酶。
本发明还提供了一种融合蛋白，包含上述的单域抗体和异源蛋白，其中异源蛋白是人的Fc。
本发明还提供了一种核酸分子，其编码上述的单域抗体或抗原结合片段，和所述的双特异性抗体，或所述的免疫偶联物，或所述的融合蛋白；所述的核酸分子，可操作地连接至启动子。
本发明还提供了一种含有所述的核酸分子的质粒，其中质粒是一种病毒质粒。
本发明还提供了一种可转化所述的核酸或所述的质粒的宿主细胞。
本发明还提供了一种药用组合物，该药用组合物含有有效预防剂量的所述的单域抗体或抗原结合片段，所述的双特异性抗体，所述的免疫偶联物，所述的融合蛋白，所述的核酸，或所述的质粒，和一种药学可接受载体。
本发明中，所述的单域抗体或抗原结合片段，所述的双特异性抗体，所述的免疫偶联物，所述的融合蛋白，或所述的药用组合物，可用于制备诊断或治疗自身免疫疾病，炎性疾病，传染病，过敏症或癌症的制剂；其中所述癌症为非霍奇金淋巴瘤，慢性淋巴细胞白血病，霍奇金氏病，实体瘤或转移瘤。
本发明中，所述的单域抗体或抗原结合片段， 所述的双特异性抗体，所述的免疫偶联物，或所述的融合蛋白，可作为表达CD16细胞的染色，或用于制备体外治疗的NK细胞分离的药物。
本发明还制备获得了针对CD16的新型全人源单域抗体；利用该抗体CDR区或部分或全基因，可在原核和真核细胞及任何表达系统中改造和生产不同形式的基因工程抗体，可用于制备临床上诊断或治疗自身免疫疾病或癌症相关疾病的制剂。
为了便于理解，以下将通过具体的附图和实施例对本发明进行详细地描述。需要特别指出的是，具体实例和附图仅是为了说明，显然本领域的普通技术人员可以根据本文说明，在本发明的范围内对本发明做出各种各样的修正和改变，这些修正和改变也纳入本发明的范围内。另外，本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
附图说明
图1. 利用多克隆噬菌体ELISA检测富集的特异性单域抗体（VH）。
图2. 利用ELISA检测特异性单域抗体（VH）与CD16的结合能力。
具体实施方式
实施例1
CD16特异性单域抗体（VH）的筛选
以人VH 3-23亚家族胚系抗体为模板，利用设计和抗体工程技术引入源于健康成年人、新生儿、癌症病人、艾滋病病人的所有抗体亚家族的重链CDR 区（CDR1, CDR2, CDR3），构建一个实际测量库容量为1500 亿（1.5×1011）的超大型全人源单域抗体库；利用此噬菌体展示单域抗体文库，针对生物素标记的CD16蛋白筛选抗体；将生物素标记的CD16蛋白固定在链亲和素包被的磁珠上，1012个噬菌体展示的抗体在常温下于1, 2, 3, 4轮分别与5, 4,2, 1微克抗原孵育两小时，每轮的筛选所用的噬菌体均为1012个；用多克隆噬菌体ELISA来检测抗体的富集；第1, 2, 3, 4轮的噬菌体与包被的蛋白孵育，用抗噬菌体的HRP耦合抗体检测噬菌体与蛋白的结合，根据多克隆噬菌体ELISA结果（如图1所示），在第3，4轮筛选后得到非常显著的富集；选用这两轮筛选获得的噬菌体，感染TG1细胞并随机挑选克隆进行单克隆噬菌体ELISA，进一步测序鉴定富集的单域抗体；
特异性单域抗体（VH）与CD16蛋白的结合能力的检测
根据测序结果选择8个克隆，它们的可溶性表达产物的制备基本按文献进行，具体为：将8个克隆的质粒转入HB2151感受态细胞，从过夜生长的氨苄平皿中挑取单个菌落，接种SB细菌培养液，在30度IPTG诱导条件下表达12-14小时；收获细菌并用Ni-TNA从中纯化出单域抗体；将CD16蛋白包被在ELISA板上，加入梯度浓度稀释的单域抗体孵育，用抗FLAG标签抗体检测单域抗体与CD16的结合能力，ELISA结果如图2所示，该8个单域抗体均能非常强的结合CD16蛋白。
SEQUENCE LISTING
<110> 复旦大学
<120> 针对CD16的全人源单域抗体、其抗原结合片段及应用
<130>
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<170> PatentIn version 3.3
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Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Ala Cys Thr Ala Thr
85 90 95
Gly Gly Thr Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Ala Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Gly Gly Ala Ala Ala Ala Thr Cys Thr Ala Thr Cys Ala Cys Ala
145 150 155 160
Gly Thr Gly Gly Ala Ala Gly Cys Ala Cys Cys Ala Ala Cys Thr Ala
165 170 175
Cys Ala Ala Cys Cys Cys Cys Thr Cys Cys Cys Thr Cys Ala Ala Gly
180 185 190
Ala Gly Thr Cys Thr Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr Cys Thr Gly
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys
260 265 270
Cys Ala Cys Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Ala Gly Ala Gly Ala Gly Thr Ala Thr Thr Gly Ala Cys Thr
290 295 300
Ala Cys Ala Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Gly
305 310 315 320
Cys Cys Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys
325 330 335
Thr Cys Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
340 345 350
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
355 360 365
Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
370 375 380
Glu Trp Ile Gly Lys Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro
385 390 395 400
Ser Leu Lys Ser Leu Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
405 410 415
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
420 425 430
Tyr Cys Ala Arg Glu Ser Ile Asp Tyr Arg Gly Gln Gly Ser Leu Val
435 440 445
Thr Val Ser Ser
450
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Gly Phe Thr Phe Ser Asn Tyr Gly
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Ile Tyr His Ser Gly Ser Thr
1 5
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Ala Arg Glu Ser Ile Asp Tyr
1 5
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Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
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Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Gly Gly Ala Ala Thr Ala Thr Cys Thr Ala Thr Cys Ala Cys Ala
145 150 155 160
Gly Thr Gly Gly Ala Ala Gly Cys Ala Cys Cys Ala Ala Cys Thr Ala
165 170 175
Cys Ala Ala Cys Cys Cys Cys Thr Cys Cys Cys Thr Cys Ala Ala Gly
180 185 190
Ala Gly Thr Cys Thr Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr Cys Thr Gly
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys
260 265 270
Cys Ala Cys Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Thr Thr Gly Ala Cys Thr
290 295 300
Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Cys
305 310 315 320
Cys Cys Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys
325 330 335
Thr Cys Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
340 345 350
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
355 360 365
Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
370 375 380
Glu Trp Ile Gly Asn Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro
385 390 395 400
Ser Leu Lys Ser Leu Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
405 410 415
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
420 425 430
Tyr Cys Ala Arg Glu Arg Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val
435 440 445
Thr Val Ser Ser
450
<210> 6
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Gly Phe Thr Phe Ser Asn Tyr Gly
1 5
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Ile Tyr His Ser Gly Ser Thr
1 5
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Ala Arg Glu Arg Ile Asp Tyr
1 5
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Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Cys Ala Ala Cys Thr Ala Thr
85 90 95
Gly Cys Thr Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Ala Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Gly Gly Thr Ala Thr Ala Thr Cys Thr Ala Thr Thr Ala Cys Ala
145 150 155 160
Gly Thr Gly Gly Ala Ala Gly Cys Ala Cys Cys Ala Ala Cys Thr Ala
165 170 175
Cys Ala Ala Cys Cys Cys Cys Thr Cys Cys Cys Thr Cys Ala Ala Gly
180 185 190
Ala Gly Thr Cys Gly Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr Cys Thr Gly
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys
260 265 270
Cys Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Ala Gly Ala Gly Thr Cys Cys Cys Thr Ala Gly Ala Cys Thr
290 295 300
Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Cys Ala Cys
305 310 315 320
Cys Cys Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys
325 330 335
Thr Cys Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
340 345 350
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
355 360 365
Ser Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
370 375 380
Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro
385 390 395 400
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
405 410 415
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr
420 425 430
Tyr Cys Ala Arg Glu Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
435 440 445
Thr Val Ser Ser
450
<210> 10
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Gly Phe Thr Phe Ser Asn Tyr Ala
1 5
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Ile Tyr Tyr Ser Gly Ser Thr
1 5
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Ala Arg Glu Ser Leu Asp Tyr
1 5
<210> 13
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Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Gly Cys Thr Ala Thr
85 90 95
Gly Gly Cys Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys
130 135 140
Thr Cys Ala Thr Cys Cys Ala Thr Thr Ala Ala Thr Ala Ala Thr Ala
145 150 155 160
Gly Thr Ala Gly Thr Ala Gly Thr Thr Ala Cys Ala Thr Ala Thr Ala
165 170 175
Cys Thr Ala Cys Cys Cys Cys Thr Ala Cys Thr Cys Cys Gly Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys
210 215 220
Cys Ala Ala Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys
245 250 255
Thr Gly Ala Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Cys Gly Cys Cys Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Gly Cys Ala Ala Gly Ala Gly Ala Cys Gly Gly Thr Cys Thr Gly Cys
290 295 300
Gly Cys Ala Thr Cys Gly Ala Cys Cys Cys Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Gly Gly Gly Ala Ala Cys Cys Cys Thr Gly Gly Thr Cys
325 330 335
Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Glu Val Gln Leu
340 345 350
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
355 360 365
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp
370 375 380
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Asn
385 390 395 400
Asn Ser Ser Ser Tyr Ile Tyr Tyr Pro Tyr Ser Val Lys Gly Arg Phe
405 410 415
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
420 425 430
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly
435 440 445
Leu Arg Ile Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
450 455 460
<210> 14
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Gly Phe Thr Phe Ser Ser Tyr Gly
1 5
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<211> 8
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Gly Phe Thr Phe Ser Ser Tyr Gly
1 5
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<211> 9
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Ala Arg Asp Gly Leu Arg Ile Asp Pro
1 5
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Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Ala Cys Thr Thr
65 70 75 80
Cys Cys Cys Gly Thr Thr Cys Ala Cys Cys Gly Cys Thr Thr Thr Gly
85 90 95
Gly Ala Ala Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Cys Cys Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Gly
130 135 140
Gly Cys Ala Ala Thr Thr Ala Thr Ala Thr Cys Ala Thr Ala Thr Gly
145 150 155 160
Ala Thr Gly Gly Ala Ala Gly Thr Ala Ala Thr Ala Ala Ala Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Cys Thr Ala Cys Thr Cys Cys Gly Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Ala Gly Ala Ala Gly Ala Cys Thr Ala Thr Thr Cys
210 215 220
Cys Ala Ala Gly Gly Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr
225 230 235 240
Cys Thr Gly Cys Ala Gly Ala Thr Gly Ala Ala Cys Cys Gly Gly Cys
245 250 255
Thr Gly Ala Gly Ala Ala Ala Cys Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Cys Gly Cys Cys Thr Thr Ala Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Gly Cys Ala Ala Ala Cys Gly Gly Thr Cys Thr Gly Gly Gly Cys Gly
290 295 300
Ala Gly Gly Ala Gly Gly Gly Thr Gly Cys Thr Thr Thr Thr Gly Ala
305 310 315 320
Thr Ala Thr Gly Thr Gly Gly Gly Gly Cys Cys Ala Cys Gly Gly Cys
325 330 335
Ala Cys Ala Ala Thr Gly Gly Gly Cys Ala Cys Cys Gly Thr Cys Thr
340 345 350
Cys Cys Thr Cys Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
355 360 365
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe
370 375 380
Pro Phe Thr Ala Leu Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys
385 390 395 400
Ala Leu Glu Trp Val Ala Ile Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
405 410 415
Tyr Ala Tyr Ser Val Lys Gly Arg Phe Thr Ile Ser Glu Asp Tyr Ser
420 425 430
Lys Asp Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Asn Glu Asp Thr
435 440 445
Ala Leu Tyr Tyr Cys Ala Asn Gly Leu Gly Glu Glu Gly Ala Phe Asp
450 455 460
Met Trp Gly His Gly Thr Met Gly Thr Val Ser Ser
465 470 475
<210> 18
<211> 8
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Asp Phe Pro Phe Thr Ala Leu Glu
1 5
<210> 19
<211> 8
<212> PRT
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<400> 19
Ile Ser Tyr Asp Gly Ser Asn Lys
1 5
<210> 20
<211> 12
<212> PRT
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<400> 20
Ala Asn Gly Leu Gly Glu Glu Gly Ala Phe Asp Met
1 5 10
<210> 21
<211> 487
<212> PRT
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<400> 21
Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Cys Cys Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Gly Gly Gly Ala Ala Ala Thr Cys Ala Cys Thr Cys Ala Cys Ala
145 150 155 160
Gly Thr Gly Gly Ala Ala Gly Cys Ala Cys Cys Ala Ala Cys Thr Ala
165 170 175
Cys Ala Ala Cys Cys Cys Gly Thr Cys Cys Cys Thr Cys Ala Ala Gly
180 185 190
Ala Gly Thr Cys Gly Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr Cys Thr Gly
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys
260 265 270
Cys Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Ala Gly Thr Gly Gly Gly Ala Cys Thr Ala Cys Ala Gly Ala
290 295 300
Cys Cys Ala Gly Cys Cys Cys Thr Cys Gly Gly Gly Ala Cys Thr Gly
305 310 315 320
Gly Thr Ala Gly Thr Thr Cys Gly Ala Thr Cys Thr Cys Thr Gly Gly
325 330 335
Gly Gly Cys Cys Gly Thr Gly Gly Ala Ala Cys Cys Cys Thr Gly Gly
340 345 350
Gly Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Glu Val
355 360 365
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
370 375 380
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met
385 390 395 400
Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Ile Gly Glu
405 410 415
Ile Thr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg
420 425 430
Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met
435 440 445
Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Val
450 455 460
Gly Leu Gln Thr Ser Pro Arg Asp Trp Phe Asp Leu Trp Gly Arg Gly
465 470 475 480
Thr Leu Gly Thr Val Ser Ser
485
<210> 22
<211> 8
<212> PRT
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Gly Phe Thr Phe Ser Asn Tyr Gly
1 5
<210> 23
<211> 7
<212> PRT
<213> n116 HCDR2
<400> 23
Ile Thr His Ser Gly Ser Thr
1 5
<210> 24
<211> 15
<212> PRT
<213> n116 HCDR3
<400> 24
Ala Arg Val Gly Leu Gln Thr Ser Pro Arg Asp Trp Phe Asp Leu
1 5 10 15
<210> 25
<211> 488
<212> PRT
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<400> 25
Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Gly Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Cys Cys Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Gly Gly Gly Ala Ala Ala Thr Cys Ala Cys Thr Cys Ala Thr Ala
145 150 155 160
Gly Thr Gly Gly Ala Ala Gly Cys Ala Cys Cys Ala Ala Cys Thr Ala
165 170 175
Cys Ala Ala Cys Cys Cys Gly Thr Cys Cys Cys Thr Cys Ala Ala Gly
180 185 190
Ala Gly Thr Cys Gly Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr Cys Thr Gly
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys
260 265 270
Cys Ala Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Ala Gly Thr Gly Gly Gly Ala Cys Thr Ala Cys Gly Gly Ala
290 295 300
Cys Cys Ala Gly Cys Cys Cys Thr Cys Gly Thr Thr Ala Cys Thr Gly
305 310 315 320
Gly Thr Ala Cys Thr Thr Cys Gly Ala Thr Cys Thr Cys Thr Gly Gly
325 330 335
Gly Gly Cys Cys Gly Thr Gly Gly Ala Ala Cys Cys Cys Thr Gly Gly
340 345 350
Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Glu Val
355 360 365
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
370 375 380
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met
385 390 395 400
Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Ile Gly Glu
405 410 415
Ile Thr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg
420 425 430
Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met
435 440 445
Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Val
450 455 460
Gly Leu Arg Thr Ser Pro Arg Tyr Trp Tyr Phe Asp Leu Trp Gly Arg
465 470 475 480
Gly Thr Leu Val Thr Val Ser Ser
485
<210> 26
<211> 8
<212> PRT
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<400> 26
Gly Phe Thr Phe Ser Ser Tyr Gly
1 5
<210> 27
<211> 7
<212> PRT
<213> n117 HCDR2
<400> 27
Ile Thr His Ser Gly Ser Thr
1 5
<210> 28
<211> 16
<212> PRT
<213> n117 HCDR3
<400> 28
Ala Arg Val Gly Leu Arg Thr Ser Pro Arg Tyr Trp Tyr Phe Asp Leu
1 5 10 15
<210> 29
<211> 452
<212> PRT
<213> n118 VH
<400> 29
Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Gly Thr Ala Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Ala Cys Thr Ala Thr
85 90 95
Gly Gly Thr Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Ala Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Gly Gly Ala Gly Thr Ala Thr Cys Thr Ala Thr Thr Ala Cys Ala
145 150 155 160
Gly Thr Gly Gly Gly Ala Gly Cys Ala Cys Cys Ala Ala Cys Thr Ala
165 170 175
Cys Ala Ala Cys Cys Cys Cys Thr Cys Cys Cys Thr Cys Ala Ala Gly
180 185 190
Ala Gly Thr Cys Thr Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr Cys Thr Gly
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys
260 265 270
Cys Ala Cys Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Ala Gly Ala Gly Ala Gly Thr Ala Thr Thr Gly Ala Cys Thr
290 295 300
Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Cys
305 310 315 320
Cys Cys Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys
325 330 335
Thr Cys Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
340 345 350
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
355 360 365
Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
370 375 380
Glu Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro
385 390 395 400
Ser Leu Lys Ser Leu Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
405 410 415
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
420 425 430
Tyr Cys Ala Arg Glu Ser Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val
435 440 445
Thr Val Ser Ser
450
<210> 30
<211> 8
<212> PRT
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<400> 30
Gly Phe Thr Phe Ser Asn Tyr Gly
1 5
<210> 31
<211> 7
<212> PRT
<213> n117 HCDR2
<400> 31
Ile Tyr Tyr Ser Gly Ser Thr
1 5
<210> 32
<211> 7
<212> PRT
<213> n117 HCDR3
<400> 32
Ala Arg Glu Ser Ile Asp Tyr
1 5
Claims (21)
1.一种针对CD16蛋白的全人源单域抗体，或抗原结合片段，其特征在于，其包含：
抗体的重链可变区含有SEQ ID NO:1的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:5的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:9的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:13的26-33位，51-58位，和/或97-105位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:17的26-33位，51-58位，和/或97-108位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:21的26-33位，51-57位，和/或96-110位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:25的26-33位，51-57位，和/或96-111位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:29的26-33位，51-57位，和/或96-102位氨基酸残基，
其中单域抗体或抗原结合片段特异性结合CD16。
2.按权利要求1所述的单域抗体或抗原结合片段，其特征在于，包含：
抗体的重链可变区含有SEQ ID NO:1的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:5的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:9的26-33位，51-57位，和/或96-102位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:13的26-33位，51-58位，和/或97-105位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:17的26-33位，51-58位，和/或97-108位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:21的26-33位，51-57位，和/或96-110位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:25的26-33位，51-57位，和/或96-111位氨基酸残基；或
抗体的重链可变区含有SEQ ID NO:29的26-33位，51-57位，和/或96-102位氨基酸残基。
3.按权利要求1或权利要求2所述的单域抗体或抗原结合片段，其特征在于，其中：
重链可变区的氨基酸序列与SEQ ID NO:1具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:5具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:9具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:13具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:17具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:21具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:25具有至少90%的相同性；或
重链可变区的氨基酸序列与SEQ ID NO:29具有至少90%的相同性。
4.按权利要求1-3中任一项所述的单域抗体或抗原结合片段，其特征在于，其中的单域抗体或抗原结合片段是全人源的。
5.按权利要求1-4中任一项所述的单域抗体或抗原结合片段，其特征在于，其中的单域抗体为VH，抗原结合片段为VH。
6.一种双特异性抗体，其特征在于，含有权利要求1-5中任一项所述的单域抗体或抗原结合片段。
7.按权利要求1-5中任一项所述的单域抗体或抗原结合片段，或权利要求6所述的双特异性抗体，其特征在于，所述的单域抗体或抗原结合片段，或所述的双特异性抗体，与一种效应分子偶联。
8.按权利要求7所述的免疫偶联物，其特征在于，其中的效应分子是可检测标记。
9.按权利要求8所述的免疫偶联物，其特征在于，其中的可检测标记是荧光标记，放射性标记，亲和素，生物素，或酶。
10.按权利要求7所述的免疫偶联物，其特征在于，其中的效应分子是毒素或化疗剂。
11.一种融合蛋白，其包含权利要求1-5中任一项所述的单域抗体和异源蛋白。
12.按权利要求11所述的融合蛋白，其中异源蛋白是人的Fc。
13.一种核酸分子，其编码权利要求1-5中任一项所述的单域抗体或抗原结合片段，权利要求6所述的双特异性抗体，或权利要求7-10中任一项所述的免疫偶联物，或权利要求11或权利要求12所述的融合蛋白。
14.按权利要求13所述的核酸分子，其特征在于，可操作地连接至启动子。
15.一种质粒，其含有权利要求13或权利要求14所述的核酸分子。
16.按权利要求15所述的质粒，其特征在于，其中质粒是一种病毒质粒。
17.一种宿主细胞，其特征在于，可转化权利要求13所述的核酸或权利要求15-16所述的质粒。
18.一种药用组合物，其特征在于，含有有效预防剂量的权利要求1-5中任一项所述的单域抗体或抗原结合片段，权利要求6所述的双特异性抗体，权利要求7-10中任一项所述的免疫偶联物，权利要求11或权利要求12所述的融合蛋白，权利要求13-14中任一项所述的核酸，或权利要求15-16中任一项所述的质粒，和一种药学可接受载体。
19.权利要求1-5中任一项所述的单域抗体或抗原结合片段，权利要求6所述的双特异性抗体，权利要求7-10中任一项所述的免疫偶联物，权利要求11或权利要求12所述的融合蛋白，或权利要求18所述的药用组合物，在制备用于诊断或治疗自身免疫疾病，炎性疾病，传染病，过敏症或癌症药物中的用途。
20.按权利要求19所述的用途，其特征在于，所述癌症为非霍奇金淋巴瘤，慢性淋巴细胞白血病，霍奇金氏病，实体瘤和转移瘤。
21.权利要求1-5中任一项所述的单域抗体或抗原结合片段，权利要求6所述的双特异性抗体，权利要求7-10中任一项所述的免疫偶联物，或权利要求11或权利要求12所述的融合蛋白，在制备作为表达CD16细胞的染色制剂，或用于体外治疗的NK细胞分离的药物中的用途。
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