具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
本发明的TCR通过以下实验环节获得:
1.特异性的CD8+T细胞克隆,获得异二聚体TCR序列;
2.针对该TCR,进行scTCR结构域的稳定性改造;
3.采用改造后的scTCR序列,进行噬菌体展示,获得高亲和力的CDR3α、CDR3β序列突变;
4.把这些突变引入到异二聚体TCR中,体外制备出相应的TCR,并测定与特异性靶点HLA*A02:01/SLLMWITQC的pMHC的亲和力,要求其KD值位于0.1~10μM区间内;
5.制备了针对一系列特异性靶点HLA-A*02:01/SLLMWITQC突变体,进一步测试其亲和力,从生化层面,获得特异性好的高亲和力异二聚体TCR突变体;
6.特异性好的高亲和力异二聚体,进一步做成慢病毒,感染CD8+T细胞,获得能够识别特异性表达HLA-A*02:01/SLLMWITQC靶点的CD8+T细胞;
7.利用改造好的CD8 T细胞,进行体外的肿瘤细胞系特异性杀伤试验,最终获得能够有效、特异杀伤肿瘤细胞的异二聚体TCR突变序列。
获得上述TCR之后,再进行动物体内杀瘤实验。
具体详见下述实施例。
以下实施例以NYc9 A5B0(NY是指NY-ESO-1这个蛋白名称,c9是指从该孔长出来的单克隆CD8+T细胞)为例详细介绍本发明TCR的制备过程。
另:以下实施例中,若没有特殊说明,所用细胞系均购自ATCC。
实施例1克隆特异性CD8+T细胞,获得异二聚体TCR序列(野生型的TCR的取得和鉴定)
T细胞克隆用到的方法、试剂和耗材,主要参考Curr.Protoc.Immunol.2002,7,1;PLoS One,2011,6,e27930;Onco Immunology 2016,5,e1175795及其引用文献。用EBV(EB病毒)转导的负载有NY-ESO-1短肽(SLLMWITQC)的B细胞(EBV-B)(J Vis Exp.2011,8,3321)(EBV病毒:ATCC产品号VR-1492)刺激HLA-A*02:01基因型健康志愿者的CD8+T细胞。其中单克隆T细胞培养方法,主要参考相关文献的工作(J Immunol Methods.2006,310,40;PLoSOne.2014,9,e110741)。用PE标记的短肽-HLA四聚体(MBL,产品号TS-M047-1)及APC标记的抗CD8抗体(Biolegend,产品号301014)分选双阳性T细胞。被SLLMWITQC短肽刺激后的T细胞扩增至5000-10000个细胞后进行分选。2~3次刺激培养和分选后,将细胞进行有限稀释至约0.5细胞/孔后培养以获取单克隆(参考Cardiff University药学系的博士论文,LissinaA.,2011,Optimisation of T cell receptor antigen recognition for targetingdisease),增殖后的单克隆T细胞用于后续四聚体染色分选。如图1所示,分选检测到的双阳性克隆细胞,用试剂盒Quick-RNATM MiniPrep(ZYMO research,产品号R1050)抽提分选到的单克隆T细胞的总RNA。mRNA进一步反转录成cDNA,采用clontech的SMART RACE cDNA扩增试剂盒,将序列克隆至pUC19(Invitrogen,产品号SD0061)上进行测序。
最终获得野生型TCR,对其进行序列鉴定、分析后,获得其α链、β链全长的组成:
α链全长:
METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCASDQDARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO.52)。
β链全长:
MDSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLGPGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSA LVLMAMVKRKDSRG(SEQ ID NO.53)。
上述α链或者β链的全长序列中:下划线标注部分为信号肽序列、无标记序列为Vα(α链的可变区;序列如SEQ ID NO.18所示)或者Vβ(β链的可变区;序列如SEQ ID NO.42所示)序列、加粗部分为Cα(α链的恒定区)或者Cβ(β链的恒定区)序列、斜体部分为胞外linker、斜体且加下划线部分为跨膜区和胞内序列。依据IMGT规则,TCRα链的FR1、CDR1(SEQID NO.71)、FR2、CDR2(SEQ ID NO.72)、FR3、CDR3(SEQ ID NO.1)以及FR4分别位于序列如SEQ ID NO.18所示的TCRα链可变区的第2~27位、28~32位、33~49位、50~56位、57~90位、91~99位以及100~109位;TCRβ链的FR1、CDR1(SEQ ID NO.73)、FR2、CDR2(SEQ IDNO.74)、FR3、CDR3(SEQ ID NO.2)以及FR4分别位于序列如SEQ ID NO.42所示的TCRβ链可变区的第1~26位、27~31位、32~48位、49~54位、55~92位、93~102位以及103~112位。
将α链全长以及β链全长的跨膜区和胞内序列(斜体且下划线标注的部分)分别去除后获得氨基酸序列如SEQ ID NO.54所示的A0和氨基酸序列如SEQ ID NO.66所述的B0(其中“A”和“B”分别代表α链和β链,“0”代表未经突变)。
实施例2针对实施例1制备得到的野生型TCR进行scTCR结构域的稳定性改造
将实施例1中获得的Vα以及Vβ(α链全长以及β链全长的可变区序列分别如SEQ IDNO.18和SEQ ID NO.42所示)利用Linker氨基酸序列连接起来获得野生型scTCR(以下简称scTCR-wt),这种结构非常不稳定,需要进行稳定性优化,才能用于亲和力优化(PNAS,1999,96,5651;Nat Biotechnol.,2000,18,754;Front.Oncol.,2015,4,1;WO2016124142)和包涵体复性(scTCR-wt的克隆、表达以及纯化过程按照本领域的常规方法)。
对scTCR-wt的β-turn结构,根据不同氨基酸侧链对β-turn不同位置的偏好性,引入有助于β-turn结构稳定性的突变。由于去除了Cα、Cβ结构域,Vα、Vβ结构域中有些位置的疏水性氨基酸暴露在表面,减少表面疏水特性的氨基酸突变,获得稳定的scTCR_X0序列。scTCR-wt和scTCR-X0直接合成基因,然后通过Nco I/Not I内切酶克隆到pET28a载体。
稳定性改造获得的scTCR_X0其突变发生于TCRα链以及TCRβ链的FR1、FR2、FR3以及FR4区,具体发生于SEQ ID NO.18所示序列,根据IMGT规则,优选位于第21、45、46、90、94、96、126以及128位中的一位或者多位;如SEQ ID NO.42所示序列中,根据IMGT规则,优选位于第5、11~15、45~47、86以及128位中的一位或者多位。由此获得A0框架突变后的可变区(序列如SEQ ID NO.30所示),以及B0框架突变后的可变区(序列如SEQ ID NO.47所示)。将A0框架突变后的可变区以及B0框架突变后的可变区以肽段Linker序列P(GGGGS)3;以下均简称为Linker序列或者Linker)相连,即得scTCR_X0[氨基酸序列如SEQ ID NO.30+Linker序列+SEQ ID NO.47所示(左为N端、右为C端;若无特殊提及,以下均为左N右C)]。
scTCR-wt和scTCR-X0的包涵体表达、蛋白复性、纯化和亲和力测试,按照实施例4、实施例5和实施例7所述完成,结果如图2A、图2B,图3A、图3B,图4A、图4B以及图5所示。
实施例3噬菌体展示
采用Nco I/Not I酶切位点,把工程改造后的scTCR的基因插入噬菌体展示载体,作为噬菌体展示文库构建模板,对可变区CDR3α/CDR3β,设计不连续的突变引物建库,电转进入TG1感受态,用于后续2-3轮噬菌体筛选。噬菌体筛选的整体过程,除了传统的分子生物学手册之外,主要参考Nature Protocols,2007,2,3001和Nat.Biotech.2005,23,349两篇文章、Cardiff University药学系的一篇博士论文(Liddy S.2013,Molecularengineering of high affinity T-cell receptors for bispecific therapeutics)。
文库筛选:文库菌接种至30~50mL包含了100μg/mL ampicillin和2%葡萄糖的2×YT培养基,接种后OD600=0.05~0.08之间,37℃,200~220rpm培养直到OD600=0.4~0.5。
按照15:1~25:1(噬菌体:细菌)的比例(摩尔比)加入辅助噬菌体,混匀后于37℃静置30分钟;室温、低速离心10分钟后弃上清,并用30~50mL加了100μg/mL ampicillin+50μg/mL kanamycin的2×YT培养基重悬,26℃,200~220rpm培养过夜。
细菌培养液4℃、高速离心菌液10min,收集上清与PEG/NaCl溶液按4:1混匀冰浴1小时,4℃,低速离心10min,收集沉淀。用10ml PBS重悬,4℃、高速离心10min,收集上清并加入2.5ml PEG/NaCl溶液,混匀后冰浴20~30min;4℃、低俗离心30分钟,沉淀用1mL PBS重悬,获得噬菌体溶液。
取适当体积的上述噬菌体溶液,加3%(w/v)牛奶溶液封闭1小时,加入适量的生物素化标记的pMHC溶液,室温孵育、反应1小时,形成噬菌体-pMHC复合体,接着加入50μl的链霉亲和素磁珠,进一步形成磁珠-噬菌体-pMHC复合体。
用磁铁吸附磁珠,充分洗涤3-5次,加入0.1mg(终浓度1mg/mL)的胰酶,室温反应30分钟,磁铁吸附后取上清感染预活化的TG1菌株,涂板、30℃倒置培养过夜。根据实验需要,重复以上流程进行第二、三轮筛选。
噬菌体ELISA实验:参考Nature Protocols,2007,2,3001的工作流程,挑取单克隆至150mL的2×YT培养基中(100μg/mL ampicillin和2%葡萄糖),37℃、200~220rpm培养过夜。每个克隆移取2~5μL菌液至新的含有150~200μL相同培养基的96孔板中,37℃、200~220rpm培养3小时。然后每孔加入50μl足够滴度的辅助噬菌体,37℃、200~220rpm培养1小时。室温,低速离心10分钟后收集沉淀,然后用200μL(加了100μg/mL ampicillin+50μg/mLkanamycin的2×YT培养基)培养基重悬;26℃、200~220rpm培养过夜。
取96孔ELISA板,每孔加入1μg的链霉亲和素,4℃冰箱静置过夜,然后PBS清洗ELISA板3次,加入0.5μg(体积浓度)生物素标记的pMHC,室温反应30分钟。加入400μL的6%牛奶PBS溶液,室温封闭1小时。
新鲜的噬菌体上清液100μL、加入等体积的牛奶溶液,室温封闭1小时后,取100μL体积的溶液,加入PBS清洗后的ELSA板中,继续室温反应1小时。
清洗ELISA板3-5次,加入稀释好的M13-HRP抗体(义翘神州,产品号11973-MM05T-H,1:10000比例稀释),室温反应30分钟,再清洗ELISA板5次,每个孔加入50μL的显色液,显色90秒,立刻终止反应,用酶标仪测定吸收值(OD450)。挑选OD450值大于0.45的单克隆,DNA测序,筛选获得相应的突变体scTCR_X1~15[其氨基酸序列分别如SEQ ID NO.30+Linker+SEQID NO.42、SEQ ID NO.30+Linker+SEQ ID NO.43、SEQ ID NO.30+Linker+SEQ ID NO.44、SEQ ID NO.30+Linker+SEQ ID NO.45、SEQ ID NO.30+Linker+SEQ ID NO.46、SEQ IDNO.31+Linker+SEQ ID NO.42、SEQ ID NO.32+Linker+SEQ ID NO.42、SEQ ID NO.33+Linker+SEQ ID NO.42、SEQ ID NO.34+Linker+SEQ ID NO.42、SEQ ID NO.35+Linker+SEQID NO.42、SEQ ID NO.36+Linker+SEQ ID NO.42、SEQ ID NO.37+Linker+SEQ ID NO.42、SEQ ID NO.38+Linker+SEQ ID NO.42、SEQ ID NO.39+Linker+SEQ ID NO.42以及SEQ IDNO.40+Linker+SEQ ID NO.42所示]。经序列分析后发现,上述突变体中的突变均发生于CDR3α以及CDR3β,具体位点如下述实施例中的表1所示。该实施例中的所述Linker同该实施例2中的Linker序列,为P(GGGGS)3。
实施例4基因克隆、包涵体表达
将实施例3中获得的突变位点如表1所示对应引入到TCR的A0和B0当中,获得氨基酸序列如SEQ ID NO.19~29所示的A1~A11的可变区,以及氨基酸序列如SEQ ID NO.43~46所示的B1~B4的可变区;并相应获得氨基酸序列分别如SEQ ID NO.55~65所示的TCRα链A1~A11、以及氨基酸序列分别如SEQ ID NO.67~70所示的TCRβ链B1~B4(“A”代表α链、“B”代表β链,带有不同的数字“A”或“B”表示含有不同突变的“α链”或“β链”;另,在下述实施例中,由TCRα链和TCRβ链组成的异二聚体简称为AmBn;其中m为0~11的整数,n为0~4的整数)。
发生于CDR3α和CDR3β上的突变位点如下表1(其中的加粗氨基酸即为突变氨基酸):
表1突变位点展示
编号 |
CDR3 |
SEQ ID NO. |
A1 |
AYDADARLM |
3 |
A2 |
AFDAHARLM |
4 |
A3 |
AYDEHARLM |
5 |
A4 |
AYDVHARLM |
6 |
A5 |
AYDQDARLM |
7 |
A6 |
AYDENARLM |
8 |
A7 |
AYDVAARLM |
9 |
A8 |
GYDQDARLM |
10 |
A9 |
SYDQEARLM |
11 |
A10 |
VYDQNARLM |
12 |
A11 |
AYDQWARLM |
13 |
B1 |
ASSLGANELF |
14 |
B2 |
ASSHGANELF |
15 |
B3 |
ASSLGSNELF |
16 |
B4 |
ASSRGSNELF |
17 |
具体的表达过程如下:
TCRα-链(A0~A11)、β-链(B0~B4)、scTCR、HLA-A*02:01(或者HLA-A*02:03、HLA-A*02:09、HLA-A*02:12、HLA-A*02:16)和β2M的基因(HLA-A*02:01、β2M和SLLMWITQC三者同时复性形成pMHC复合体;其中HLA-A*02:01的UniProt ID是为P01892,HLA-A*02:03、HLA-A*02:09、HLA-A*02:12、HLA-A*02:16是HLA-A*02:01的多态突变体、β2M的UniProt ID是P61769)采用Nco I/Not I酶切位点克隆到pET28a的载体(购自Novagen)中,分别转化E.coli BL21(DE3)(购自NEB公司),挑取单克隆到LB培养基中,37℃振荡培养至OD600=0.6~0.8,然后加入IPTG至终浓度为0.8mM,37℃继续培养3小时。6000rpm离心10分钟,收集菌体,放入-20℃保存。
实施例5包涵体纯化、复性和纯化
用裂解液(0.5%TritonX 100的PBS)重悬菌体,超声破碎后,12000rpm高速离心20分钟。弃上清,用裂解液重悬沉淀直至无肉眼可见的颗粒,再高速离心10分钟,重复以上操作2-3次,用6M盐酸胍溶液溶解沉淀,高速离心10分钟,收集上清,上清即为纯化后的包涵体,上清取1μL进行SDS-PAGE电泳,图6显示包涵体的纯度符合要求。定量、分装,-80℃冻存。
将20mg TCRα链和15mgβ链(实施例4制备得到)分别稀释在5mL的6M盐酸胍溶液中。将TCRα链、TCRβ链依次缓慢加入到预冷的复性缓冲液(Science 1996,274,209;J.Mol.Biol.1999,285,1831;Protein Eng.2003,16,707),4℃下持续搅拌、混合30分钟。然后将其加入透析袋中,放入10倍体积预冷的去离子水中,搅拌透析8-12小时。在预冷的透析外液(pH 8.1,20mM Tris-HCl)、4℃透析8小时,重复2-3次。
将透析袋中的溶液倒出,高速离心10分钟去除沉淀和气泡,通过HiTrap Q HP(5ml)进行阴离子交换层析,0-2M NaCl,20mM Tris pH 8.1线性洗脱。收集洗脱峰,合并浓缩含有目标蛋白组分的洗脱峰,在非还原性SDS-PAGE电泳中,48kD附近有一条带,显示为NYc9-A5B0,但是纯度还不满足要求,需进一步纯化。浓缩后的蛋白样品,用superdex 75 10/300进行分子筛层析。非还原性SDS-PAGE电泳检测,48kD附近有一高浓度条带,采用还原性SDS-PAGE电泳检测,有两条带,分别是α-链和β-链,纯度达90%左右。具体如图7A、图7B以及图8A、图8B所示(鉴于A0~A11的分子量相同,B1~B4的分子量相同,此处仅以A5和B0为例显示其纯化结果)。
实施例6生物素化抗原肽-MHC(pMHC)制备
pMHC的复性和纯化,按照NIH Tetramer Core Facility的方法进行制备(http://tetramer.yerkes.emory.edu/support/protocols)。现以HLA-A*02:01/SLLMWITQC为实施代表例,按照在线protocols所述,将多肽溶液与β2M,HLA-A*02:01的包涵体溶液依次加入复性缓冲液(0.1M Tris-HCl,0.4M L-arginine,2mM EDTA;0.5mM氧化性谷胱甘肽和5mM还原性谷胱甘肽,0.2mM PMSF),4℃搅拌过夜,第二天早上和晚上分别再加入同量的HLA-A*02:01的包涵体溶液,4℃搅拌1~3天。然后在10倍体积透析液(pH 8.1,20mM Tris-HCl)中透析3次。将透析后的蛋白样品使用HiTrap Q HP(5ml)进行阴离子交换层析,采用0~2MNaCl,20mM Tris pH 8.1溶液线性洗脱,收集洗脱峰,采用SDS-PAGE电泳分析,有较纯HLA-A*02:01和β2M两条带,而SLLMWITQC分子量太小,胶图看不到条带。合并浓缩含有pMHC组分的洗脱峰,经凝胶过滤层析(Superdex 75 10/300)进一步纯化,然后用SDS-PAGE电泳检测,从电泳图上可知,得到纯度更好的pMHC复合体。用重组酶BirA(BPS Bioscience产品,产品号:70031)进行生物素化(Protein Expr.Purif.2012,82,162;J.Bacteriol.2012,194,1113.),反应体系按照NIH Tetramer Core Facility的方法进行制备、Gel Shift纯度鉴定。从Gel Shift电泳图来看,纯度符合要求。结果详见图9A、图9B,图10A、图10B以及图11。
实施例7亲和力测试
Octet是一种采用SPR技术检测亲和力的仪器,根据基于光纤生物传感器的生物膜层光学干涉技术,检测相互作用分子间的动力学参数,进行动力学和亲和力分析,计算出结合解离常数。在本实验中,我们采用SA传感器固定生物素化的pMHC,检测其与不同TCR的结合解离常数,计算出KD值。以NYc9 A5B0作为实施代表例,测试HLA-A*02:01/SLLMWITQC的亲和力,详见图12。
以下表2是对NYc9的TCR突变体和HLA-A*02:0x/SLLMWITQC结合亲和力的一个整理总结。从结果来看,大多数NYc9突变体,结合HLA-A*02:0x/SLLMWITQC的KD位于0.1~10μM之间,较佳地位于0.39~9.3μM之间,更佳地位于0.81~3.2μM之间,所述的x为1、3、9、12和16。
需说明的是:如本领域人员所知,SPR技术是当前测定亲和力最常有、可靠的方法之一,但是涉及到蛋白定量、芯片新旧程度、仪器状态等,不同批次间的实验,会有一定的误差,误差值甚至可达3~5倍;而本发明为使用了同一蛋白定量、同一芯片以及同一仪器所进行的同批次实验,因此各数据之间可用于进行亲和力大小的比较,但具体数值并不构成对本发明保护范围的限制。
表2下划线为突变氨基酸
实施例8慢病毒制备和感染CD8+T细胞
(a)野生型和突变型NY-ESO-1TCR慢病毒包装。采用第三代慢病毒包装系统(Invitrogen,pLenti6/V5 Directional TOPOTM Cloning Kit,产品号K495510)包装含有编码所需TCR的基因的慢病毒,为了降低与CD8+T细胞本身TCR的错配问题,参考过去相关文献工作,引入了v-Fos/v-Jun形成的Coiled-coil结构,用于促进TCR的α链和β链配对折叠(PNAS,1994,91,11408;Mol.Ther.Oncolytics,2017,5,105)。还需要引入P2A的自水解序列(Nat.Biotech.2004,22,589;Gene Ther.,2008,15,1411;J Immunother.,2008,31,830;),促进外源转入的αβTCR多顺反子的同步表达、折叠。把TCRα链的C端把v-Jun相关序列连接起来,形成了TCRα链+v-Jun相关序列,简称TRAJun,同理,把TCRβ链+v-Fos相关序列连起来,简称TRBFos。
v-Jun相关序列:SGSGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY v-Fos相关序列:SGSGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY P2A相关序列:SGRAKRSGSGATNFSLLKQAGDVEENPGP
其中下划线分别为v-Jun、v-Fos和P2A的序列,SG、SGSG是连接序列,RAKR是Furin酶切位点(J Biol.Chem.1999,274,23229)。例如,插入包含v-Fos/v-Jun和P2A的相关序列后,需要表达的A1B0的全氨基酸序列为:SEQ ID NO.19+v-Jun相关序列+P2A相关序列+SEQID NO.42+v-Fos相关序列,表达A1B0的慢病毒载体序列详见图26B;插入包含c-Fos/c-Jun和P2A的相关序列后,需要表达的A0B1的慢病毒载体序列为:SEQ ID NO.18+v-Jun相关序列+P2A相关序列+SEQ ID NO.43+v-Fos相关序列,表达A0B1的慢病毒载体详见图26M;分别表达A0B0、A1B0、A2B0、A3B0、A4B0、A5B0、A6B0、A7B0、A8B0、A9B0、A10B0、A11B0、A0B1、A0B2、A0B3以及A0B4时的慢病毒载体序列可详见于图26A~图26P。
具体地讲,将包装野生型和突变型pLenti6-NY-ESO-1TRAJun-2A-TRBFos和pLenti6-eGFP假病毒,与包装质粒pMDLg/pRRE(addgene,产品号k12251),pRSV-REV(addgene,产品号12253)和pMD2.G(addgene,产品号12259)按3:2:2:1的比例(详见产品手册)混匀,瞬时转染处于对数生长期的293T细胞(购自ATCC,产品号CRL-3216)。转染试剂PEI-MAX(购自Polyscience,产品号23966-1)与质粒的使用比例是2:1(体积质量比),具体操作步骤按照说明书进行。
第3和第4天收集含有包装的慢病毒的培养基上清,浓缩。把所收集到的培养基上清用50kD分子量截留的浓缩管(Merck Millipore)进行浓缩,至终体积为1毫升,等份分装后-80℃冻存。取假病毒样品进行病毒滴度测定,步骤参照p24ELISA(Clontech,产品号632200)试剂盒说明书。作为对照用,同时也包转pLenti6-eGFP的假病毒。
(b)用含有NY-ESO-1特异性T细胞受体基因的慢病毒转导原代CD8+T细胞
CD8+T细胞分离与刺激扩增、试剂使用按照参考文献(J Immunol Methods.2006,310,40;J Transl.Med.2010,8,104;Nat Protoc.2014,9,950)进行。从健康志愿者的血液中,通过负分离法富集CD8+T细胞(抗体偶联磁珠购自Miltenyi Biotec),抗体-磁珠使用方法按照产品说明书进行,CD8+T细胞分离效果可以达到90%以上。在含有50IU/mL IL-2(Peprotech,产品号AF-200-02)和10ng/mL IL-7(Peprotech,产品号AF-200-07)的RPMI-1640完全培养基(10%FBS)中,将CD8+T细胞与预洗涤的抗CD3/CD28抗体-包被小珠(LifeTechnologies,产品号11452D)共孵育过夜刺激,细胞:珠=1:1。
根据病毒滴度,按MOI=10的比例,加入浓缩好的慢病毒,32℃,900g离心感染1小时。去除慢病毒感染液,参考文献及其相关工作(J Immunol Methods,1990,128,189),以包含50IU/mL IL-2和10ng/mL IL-7的RPMI-1640完全培养基重悬细胞,37℃/5%CO2条件下培养。转导第3天通过流式细胞术分析细胞感染效率,转导第5天开始用于功能试验(例如,IFN-γ释放的ELISPOT和非放射性细胞毒性检测)。
流式细胞术分析CD8+T细胞中TCR的转导效率(PE标记TCR,APC标记CD8),结果详见图13,具体参考了文章(Blood,2010,115,3718)的操作方案。
实施例9验证NY-ESO-1特异性TCR功能-ELISPOT方案检测T2细胞多肽负载的INF-γ释放
本实验方案以IFN-γ的产量作为T细胞激活的标识,检测TCR-转导的T细胞对靶细胞特异性激活反应,实验方案参考文献(PNAS,2011,108,2991)进行。
本试验的靶细胞是T2细胞,效应细胞是实施例8中经流式细胞术分析并表达NY-ESO-1TCR的CD8+T细胞,并以同一志愿者的CD8+T细胞作为阴性对照效应细胞。将CD8+T细胞以2×所需终浓度重悬在试验培养基(10%FBS的RPMI 1640)中。
按照生产商提供的说明书,准备PVDF ELISPOT 96孔板(Merck Millipore,产品号MSIPS4510):用无菌PBS按1:200比例稀释抗人IFN-γ捕捉抗体(人IFN-γELISPOT PVDF-酶试剂盒,BD公司,产品号551849),4℃孵育过夜。洗涤除去多余的捕捉抗体后,以10%FBS的PBS在室温下封闭孔板2小时。
然后依次将试验的诸组分加入ELISPOT孔板:1.10000个T2细胞/孔;2.1000个NY-ESO-1TCR CD8+双阳性T细胞,或者阴性对照CD8+T细胞;3.20微升浓度为10μM的SLLMWITQC短肽(阳性多肽)、三条非特异性短肽溶液VLDGLDVLL(阴性多肽1)、GLYDGMEHL(阴性多肽2)、TIHDIILECV(阴性多肽3),终浓度为1μM。所有实验组一式三份。
温育孔板过夜(37℃/5%CO2),然后按照人IFN-γELISPOT PVDF-酶试剂盒使用说明操作,弃培养基,用双蒸水及洗涤缓冲液(0.01M PBS/0.05%Tween20)洗涤后,以10%FBS的PBS稀释检测一抗,室温下温育孔板2小时,洗涤,再以10%FBS的PBS稀释,在室温下温育1小时。经洗涤缓冲液洗涤3次,PBS洗涤2次后,加入试剂盒提供的BCIP/NBT溶液100微升/孔进行显影5-15分钟。去除BCIP/NBT溶液并用双蒸水冲洗孔板以中止显影反应,在室温下干燥孔板直至每个孔完全干燥。结果见图14,NYc9A2B0、A3B0和A5B0只有在T2细胞负载了SLLMWITQC的阳性多肽之后,才有INF-γ释放信号,而不负载多肽、负载了VLDGLDVLL(阴性多肽1)、GLYDGMEHL(阴性多肽2)、TIHDIILECV(阴性多肽3),都没有刚查到明显的INF-γ释放信号,说明NYc9 A2B0、A3B0和A5B0特异性识别HLA-A*02:01/SLLMWITQC。
实施例10验证NY-ESO-1特异性TCR功能-肿瘤细胞系LDH特异性杀伤
本试验是51Cr释放细胞毒性试验的比色替代试验,定量测定细胞裂解后释放的乳酸脱氢酶(LDH),实验方案参考文献(Eur.J Immunol.1993,23,3217)进行。采用30分钟偶联的酶反应来检测释放在培养基中的LDH,在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板仪收集490nm可见光吸光值数据。本试验采用A375、U266、293T和NCI-H1299四株细胞系为靶细胞,每孔接种1.5×104个细胞。效应细胞(T细胞)是实施例8中经流式细胞术分析表达NY-ESO-1特异性TCR的CD8+T细胞。效应细胞与靶细胞之比采用10:1/5:1/2.5:1/1.25:1/0.625:1。设同源CD8+T细胞加靶细胞为对照组(5:1)。
依次将试验的诸组分加入微孔圆底96孔组织培养板:1.100μL靶细胞(如上所述制备,1.5×104个靶细胞/孔);2.100μL效应细胞(如上所述制备)。增添对照组:1、效应细胞自发释放:仅有100μL效应细胞。2、靶细胞自发释放:仅有100μL靶细胞。3、靶细胞最大释放:仅有100μL靶细胞(实验时需额外加入裂解液)。4、培养基对照:仅有200μL培养基。5、体积校正孔:仅有200μL培养基。所有实验组一式三份,终体积为200μL(不够的用培养基补足)。
实验使用CytoTox
非放射性细胞毒性检测试剂盒(Promega,G1780,含有底物混合物、试验缓冲液、裂解溶液和终止缓冲液)。收集所有孔上清前,将靶细胞最大释放对照孔和体积校正孔加入20μL裂解溶液,在37℃放置30分钟,以使靶细胞全部裂解。
37℃温育混合细胞24小时后,250g离心平板4分钟,将试验平板各孔的50μL上清转移至96孔免疫平板Maxisorb板的相应孔。每孔加入50μL底物混合物,避光室温温育30分钟。将50μL终止溶液加入平板各孔以终止反应。加入终止溶液后1小时内读取490nm的吸光值。
计算结果:从实验组、靶细胞自发释放组和效应细胞自释放组的所有吸光值中扣除培养基背景的吸光值。将上述获得的经过校正的值带入下面公式,计算每个效靶比所产生的细胞毒性百分比。细胞毒性(%)=100×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)。
结果显示,表达TCR突变体A3B0和A5B0的CD8+T细胞,选择性杀伤HLA-A*02:01及NY-ESO-1双阳性的人黑色素瘤细胞A375及人髓系恶性白血病细胞U266B1和IM9,而对单阳性的人肾上皮细胞293T及人肺癌细胞NCI-H1299不具有特异性的杀伤作用,与阴性对照GFPT细胞相似。
基于上述实验,针对A3B0、A5B0的LDH杀伤实验做了优化(其他条件不变,优化E:T为2.5:1),并引入IM9的阳性细胞系,实验结果见图15。根据LDH的结果,A3B0、A5B0对肿瘤细胞的特异性杀伤活性与阳性对照1G4具有可比性;具体地,A3B0、A5B0比阳性对照1G4更好,尤其是A5B0对A375、IM9的杀伤效果,高出10%左右。
实施例11验证NY-ESO-1特异性TCR功能-A375黑色素瘤异源移植实验
实验用NOD/SCID小鼠,雌性,4周龄。动物购入后,被安置在SPF级别动物饲养中心,每笼5只。动物饲养室内温度保持在20±2℃,每小时更换空气15次,每天光照时间平分(12小时光线:12小时黑暗),相对湿度保持在50%~55%。鼠笼内置硬木屑,各组小鼠统一喂标准饲料和清洁饮用纯净水。各组小鼠均适应性饲养1周后才进行后续实验。
小鼠随机分为阴性对照组(GFP-T细胞)、阳性对照组(1G4 TCR-T细胞;其中的1G4为文献J.Immunol.2008,180,6116.中提及的α95-LY突变体,其序列由生物公司合成)、实验组(A3B0 TCR-T细胞和A5B0 TCR-T细胞),每组各5只,合计20只小鼠,小鼠剪耳做切口标记。实验采取皮下接种细胞的方式,建立肿瘤动物模型,具体而言,每只小鼠右后肢背部剃毛并接种5×106的A375细胞(人黑色素瘤细胞)及1.5×107T细胞的混合液(细胞收集于PBS缓冲液中),每只小鼠接种细胞总体积为200μL,接种细胞后,每只小鼠腹腔注射人重组IL-2 100μL(储存液浓度50万单位/mL),连续注射5天。接种十天后,开始测量肿瘤体积,此后每隔两天测量一次肿瘤体积,记录肿瘤长短径,计算体积大小,绘制肿瘤生长曲线。肿瘤体积计算公式为V=1/6×π×a×b2(a为长径,b为短径)。一个月后CO2安乐处死小鼠。
各组小鼠负荷肿瘤生长情况见图16。与阴性对照组GFP-T细胞相比,1G4 TCR-T细胞、A3B0 TCR-T细胞及A5B0 TCR-T细胞均能有效杀伤肿瘤细胞A375,抑制肿瘤的生长,肿瘤生长曲线随时间的变化均无明显变化,详见图17(鉴于A3B0 TCR-T细胞与A5B0 TCR-T细胞效果相当,以下实施例中以A5B0为例进行后续实验)。
实施例12验证NY-ESO-1特异性TCR功能-健康人PBMC特异性INF-γ释放
为排查NYC9高亲和力突变体的安全性,采用实施例9中使用的INF-γ释放实验,对40例健康人PBMC进行安全性排查。10例健康人的PBMC包含HLA-A*02:01分型,30例健康人的PBMC不包含HLA-A*02:01分型。从INF-γ释放反应的结果,NYc9 A5B0TCR-T细胞不与来自40例健康人的PBMC发生明显反应,不论10例PBMC的HLA分型是A*02:01(NYc9 A5B0对10例来自健康人的HLA-A*02:01分型PBMC的INF-γ释放数据,其反应强度与阴性、阳性对照相当;详见图18),还是30例PBMC的非HLA-A*02:01分型(NYc9 A5B0对30例来自健康人的非HLA-A*02:01分型PBMC的INF-γ释放数据,其反应强度与阴性、阳性对照相当;详见图19)。
实施例13验证NY-ESO-1特异性TCR功能-健康人原代细胞特异性INF-γ释放
为排查NYC9高亲和力突变体的安全性,采用实施例9使用的INF-γ释放实验,对5例重要脏器的原代细胞进行安全性排查。其中CCC-HEK-1、MRC-5、CCC-HEH-2、CCC-HEL-1、CCC-HPF-1都是来自胚胎的原代细胞,购买自国家实验细胞资源共享平台,MRC-5来自ATCC。从INF-γ释放反应的结果,NYc9 A5B0 TCR-T细胞的反应强度与阴性、阳性对照相当,不与这些重要器官的原代细胞发生明显反应(详见图20)。
实施例14验证NY-ESO-1特异性TCR功能-多个HLA-A*02:0x分型功能验证
为排查NYC9高亲和力突变体对多个HLA-A*02:0x分型的兼容性,通过携带HLA-A02:0x基因的慢病毒感染HLA-A*02阴性、NY-ESO-1阳性的NCI-H1299肿瘤细胞系。然后采用实施例9中使用的INF-γ释放实验,对感染了不同HLA-A*02:0x的NC-H1299进行INF-γ释放实验,评价NYc9 A5B0高亲和力突变体对多个HLA-A*02:0x分型的活性。图21显示NYc9 A5B0对HLA-A*02:03有明显活性,对HLA-A*02:12活性较好,对HLA-A*02:01、HLA-A*02:09、HLA-A*02:16活性相当。
实施例15NY-ESO-1特异性TCR功能-多个HLA-A*02:0x分型安全性验证
为排查NYc9 A5B0高亲和力突变体对多个HLA-A*02:0x分型的安全性,采用HLA-A02:0x基因的慢病毒感染多种人原代细胞,比如人星形胶质细胞(HA)、人肺成纤维细胞(HLF)、人胚胎肝细胞(CCC-HEL)和人胚胎肺细胞(CCC-HPF),然后采用实施例9中使用的INF-γ释放实验,对感染了不同HLA-A*02:0x的人原代细胞进行INF-γ释放实验,评价NYc9A5B0高亲和力突变体对多个HLA-A*02:0x分型的安全性。图22、图23、图24和图25分别显示了NYc9 A5B0对感染了多种HLA-A*02:0x分型的人星形胶质细胞(HA)、人肺成纤维细胞(HLF)、人胚胎肝细胞(CCC-HEL)和人胚胎肺细胞(CCC-HPF)的INF-γ释放实验结果,显示NYc9 A5B0对多种HLA-A*02:0x分型的识别是安全的。
SEQUENCE LISTING
<110> 深圳普瑞金生物药业股份有限公司
<120> 一种TCR及其应用
<130> P19012717C
<140> 2019108125559
<141> 2019-08-30
<160> 74
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> Homo sapiens
<400> 1
Ala Ser Asp Gln Asp Ala Arg Leu Met
1 5
<210> 2
<211> 10
<212> PRT
<213> Homo sapiens
<400> 2
Ala Ser Ser Leu Gly Pro Gly Glu Leu Phe
1 5 10
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A1 CDR3
<400> 3
Ala Tyr Asp Ala Asp Ala Arg Leu Met
1 5
<210> 4
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A2 CDR3
<400> 4
Ala Phe Asp Ala His Ala Arg Leu Met
1 5
<210> 5
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A3 CDR3
<400> 5
Ala Tyr Asp Glu His Ala Arg Leu Met
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A4 CDR3
<400> 6
Ala Tyr Asp Val His Ala Arg Leu Met
1 5
<210> 7
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A5 CDR3
<400> 7
Ala Tyr Asp Gln Asp Ala Arg Leu Met
1 5
<210> 8
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A6 CDR3
<400> 8
Ala Tyr Asp Glu Asn Ala Arg Leu Met
1 5
<210> 9
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A7 CDR3
<400> 9
Ala Tyr Asp Val Ala Ala Arg Leu Met
1 5
<210> 10
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A8 CDR3
<400> 10
Gly Tyr Asp Gln Asp Ala Arg Leu Met
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A9 CDR3
<400> 11
Ser Tyr Asp Gln Glu Ala Arg Leu Met
1 5
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A10 CDR3
<400> 12
Val Tyr Asp Gln Asn Ala Arg Leu Met
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> A11 CDR3
<400> 13
Ala Tyr Asp Gln Trp Ala Arg Leu Met
1 5
<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> B1 CDR3
<400> 14
Ala Ser Ser Leu Gly Ala Asn Glu Leu Phe
1 5 10
<210> 15
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> B2 CDR3
<400> 15
Ala Ser Ser His Gly Ala Asn Glu Leu Phe
1 5 10
<210> 16
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> B3 CDR3
<400> 16
Ala Ser Ser Leu Gly Ser Asn Glu Leu Phe
1 5 10
<210> 17
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> B4 CDR3
<400> 17
Ala Ser Ser Arg Gly Ser Asn Glu Leu Phe
1 5 10
<210> 18
<211> 109
<212> PRT
<213> Homo sapiens
<400> 18
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Ser Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 19
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A1
<400> 19
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Ala Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 20
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A2
<400> 20
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Phe Asp Ala His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 21
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A3
<400> 21
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Glu His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 22
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A4
<400> 22
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Val His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 23
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A5
<400> 23
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 24
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A6
<400> 24
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Glu Asn Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 25
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A7
<400> 25
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Val Ala Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 26
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A8
<400> 26
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Gly Tyr Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 27
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A9
<400> 27
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ser Tyr Asp Gln Glu Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 28
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A10
<400> 28
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Val Tyr Asp Gln Asn Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 29
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A11
<400> 29
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Gln Trp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
100 105
<210> 30
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A0
<400> 30
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Ser Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 31
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A1
<400> 31
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Ala Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 32
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A2
<400> 32
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Phe Asp Ala His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 33
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A3
<400> 33
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Glu His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 34
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A4
<400> 34
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Val His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 35
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A5
<400> 35
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 36
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A6
<400> 36
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Glu Asn Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 37
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A7
<400> 37
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Val Ala Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 38
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A8
<400> 38
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Gly Tyr Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 39
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A9
<400> 39
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ser Tyr Asp Gln Glu Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 40
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A10
<400> 40
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Val Tyr Asp Gln Asn Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 41
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> A11
<400> 41
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Thr
65 70 75 80
Arg Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Gln Trp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Asn
100 105
<210> 42
<211> 112
<212> PRT
<213> Homo sapiens
<400> 42
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Pro Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
<210> 43
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B1
<400> 43
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ala Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
<210> 44
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B2
<400> 44
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser His
85 90 95
Gly Ala Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
<210> 45
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B3
<400> 45
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ser Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
<210> 46
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B4
<400> 46
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Arg
85 90 95
Gly Ser Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
<210> 47
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B0
<400> 47
Asp Ala Gly Val Thr Gln Ser Pro Arg His Ile Thr Val Pro Gln Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser His Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Pro Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Asn
100 105 110
<210> 48
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B1
<400> 48
Asp Ala Gly Val Thr Gln Ser Pro Arg His Ile Thr Val Pro Gln Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser His Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ala Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Asn
100 105 110
<210> 49
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B2
<400> 49
Asp Ala Gly Val Thr Gln Ser Pro Arg His Ile Thr Val Pro Gln Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser His Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser His
85 90 95
Gly Ala Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Asn
100 105 110
<210> 50
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B3
<400> 50
Asp Ala Gly Val Thr Gln Ser Pro Arg His Ile Thr Val Pro Gln Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser His Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ser Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Asn
100 105 110
<210> 51
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> B4
<400> 51
Asp Ala Gly Val Thr Gln Ser Pro Arg His Ile Thr Val Pro Gln Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser His Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Arg
85 90 95
Gly Ser Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Asn
100 105 110
<210> 52
<211> 270
<212> PRT
<213> Homo sapiens
<400> 52
Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser
20 25 30
Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser
35 40 45
Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val
50 55 60
His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg
65 70 75 80
Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile
85 90 95
Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Ser Asp
100 105 110
Gln Asp Ala Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys
115 120 125
Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
130 135 140
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
145 150 155 160
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
165 170 175
Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
180 185 190
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
195 200 205
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys
210 215 220
Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
225 230 235 240
Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val
245 250 255
Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265 270
<210> 53
<211> 310
<212> PRT
<213> Homo sapiens
<400> 53
Met Asp Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala
1 5 10 15
Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr
20 25 30
Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His
35 40 45
Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu
50 55 60
Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro
65 70 75 80
Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu
85 90 95
Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala
100 105 110
Ser Ser Leu Gly Pro Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu
115 120 125
Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
130 135 140
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
145 150 155 160
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
165 170 175
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
180 185 190
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
195 200 205
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
210 215 220
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
225 230 235 240
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
245 250 255
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
260 265 270
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
275 280 285
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
290 295 300
Arg Lys Asp Ser Arg Gly
305 310
<210> 54
<211> 221
<212> PRT
<213> Homo sapiens
<400> 54
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Ser Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 55
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A1
<400> 55
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Ala Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 56
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A2
<400> 56
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Phe Asp Ala His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 57
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A3
<400> 57
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Glu His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 58
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A4
<400> 58
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Val His Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 59
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A5
<400> 59
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 60
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A6
<400> 60
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Glu Asn Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 61
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A7
<400> 61
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Val Ala Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 62
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A8
<400> 62
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Gly Tyr Asp Gln Asp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 63
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A9
<400> 63
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ser Tyr Asp Gln Glu Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 64
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A10
<400> 64
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Val Tyr Asp Gln Asn Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 65
<211> 221
<212> PRT
<213> Artificial Sequence
<220>
<223> A11
<400> 65
Met Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu
1 5 10 15
Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn
20 25 30
Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile
35 40 45
Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val
50 55 60
Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser
65 70 75 80
Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Tyr Asp Gln Trp Ala
85 90 95
Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
<210> 66
<211> 259
<212> PRT
<213> Homo sapiens
<400> 66
Met Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met
1 5 10 15
Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr
20 25 30
Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile
35 40 45
Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp
50 55 60
Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile
65 70 75 80
Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser
85 90 95
Leu Gly Pro Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr
<210> 67
<211> 259
<212> PRT
<213> Artificial Sequence
<220>
<223> B1
<400> 67
Met Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met
1 5 10 15
Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr
20 25 30
Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile
35 40 45
Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp
50 55 60
Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile
65 70 75 80
Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser
85 90 95
Leu Gly Ala Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr
<210> 68
<211> 259
<212> PRT
<213> Artificial Sequence
<220>
<223> B2
<400> 68
Met Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met
1 5 10 15
Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr
20 25 30
Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile
35 40 45
Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp
50 55 60
Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile
65 70 75 80
Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser
85 90 95
His Gly Ala Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr
<210> 69
<211> 259
<212> PRT
<213> Artificial Sequence
<220>
<223> B3
<400> 69
Met Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met
1 5 10 15
Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr
20 25 30
Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile
35 40 45
Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp
50 55 60
Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile
65 70 75 80
Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser
85 90 95
Leu Gly Ser Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr
<210> 70
<211> 259
<212> PRT
<213> Artificial Sequence
<220>
<223> B4
<400> 70
Met Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met
1 5 10 15
Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr
20 25 30
Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile
35 40 45
Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp
50 55 60
Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile
65 70 75 80
Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser
85 90 95
Arg Gly Ser Asn Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr
<210> 71
<211> 5
<212> PRT
<213> Homo sapiens
<400> 71
Thr Ser Ile Asn Asn
1 5
<210> 72
<211> 7
<212> PRT
<213> Homo sapiens
<400> 72
Ile Arg Ser Asn Glu Arg Glu
1 5
<210> 73
<211> 5
<212> PRT
<213> Homo sapiens
<400> 73
Ser Gly His Asp Tyr
1 5
<210> 74
<211> 6
<212> PRT
<213> Homo sapiens
<400> 74
Phe Asn Asn Asn Val Pro
1 5