CN102439034B - 双功能多肽 - Google Patents
双功能多肽 Download PDFInfo
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- CN102439034B CN102439034B CN201080022523.6A CN201080022523A CN102439034B CN 102439034 B CN102439034 B CN 102439034B CN 201080022523 A CN201080022523 A CN 201080022523A CN 102439034 B CN102439034 B CN 102439034B
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Abstract
一种双功能多肽,其含有肽-MHC表位的特异性结合伴侣如抗体或T细胞受体(“TCR”),以及免疫效应子如抗体或细胞因子,所述免疫效应子部分与所述肽-MHC结合部分的N-末端结合。
Description
本发明涉及双功能多肽,其含有肽-MHC表位的结合伴侣如抗体或T细胞受体(“TCR”),以及免疫效应子如抗体或细胞因子,所述免疫效应子部分与所述肽-MHC结合部分的N-末端结合。
发明背景
TCR介导识别特异性主要组织相容性复合抗体(MHC)-肽复合体(“pMHC复合体”),该复合体作为表位存在于抗原呈递细胞(APC)上,TCR介导T细胞对这类pMHC表位的识别。因此TCR对于免疫系统细胞组分发挥作用很关键。
抗体也是已知的,其特异性结合抗原呈递细胞所呈递的pMHC表位(参见例如:Neethling FA.等,Vaccine(2008)26(25):3092-102)。有特异性结合pMHC表位的抗原结合片段(Fab)抗体(参见例如:Chames P.等,Proc NatlAcad Sci U S A(2000)97(14):7969-74;Willemsen RA.等,J Immunol(2005)174(12):7853-8;Willemsen R.等,Cytometry A(2008)73(11):1093-9)或单链抗体片段(scFv)(参见例如:Denkberg G.等,J Immunol(2003)171(5):2197-207;Marget M.等,Mol Immunol(2005)42(5):643-9)。
天然TCR是免疫球蛋白超家族的异二聚体细胞表面蛋白,和参与介导信号转导的CD3复合体的非变异蛋白相关联。TCR以αβ和γδ形式存在,这两种形式结构相似但在解剖学位置与可能功能上差异显著。MHC I类和II类配体也是免疫球蛋白超家族蛋白,但专用于抗原呈递,其中高度多态性的肽结合位点使它们能在APC细胞表面呈递各种短肽片段。
天然异二聚体αβTCR的胞外部分由两个多肽(α和β链)组成,每个多肽含近膜恒定区和远膜可变区。恒定区和可变区各包括链内的二硫键。可变区含有与抗体的互补决定区(CDR)类似的高度多态性环。αβTCR的CDR3与MHC呈递的肽相互作用,而αβTCR的CDR 1和2与所述肽和MHC相互作用。TCR序列的多样性由连接的可变区(V)、多样区(D)、连接区(J)和恒定区(C)基因的体细胞重排产生。
功能性α链多肽由重排的V-J-C区形成,而β链由V-D-J-C区组成。胞外恒定区具有近膜区和免疫球蛋白区。有单链α链恒定区,称为TRAC。β链的恒定区由两种不同β恒定区中的一种组成,这两种恒定区称为TRBC1和TRBC2(IMGT命名)。这些β恒定区之间有四个氨基酸变化。这些变化都在TRBC1和TRBC2的外显子1内:N4K5->K4N5和F37->Y(IMGT编号,区分TRBC1->TRBC2),两种TCRβ链恒定区之间最后的氨基酸变化在TRBC1和TRBC2的外显子3内:V1->E。
目前已设计了多种构建体用于生产重组TCR。这些构建体分成两大类,单链TCR和二聚体TCR。单链TCR(scTCR)是由单个氨基酸链组成的人工构建体,和天然异二聚体TCR类似与MHC-肽复合体结合。scTCR可由TCRα和β可变区(分别为Vα和Vβ)以及TCRα和β恒定区(分别为Cα和Cβ)的组合组成,通过接头序列(L)以多种可能的取向连接,例如但不限于下列连接:Vα-L-Vβ、Vβ-L-Vα、Vα-Cα-L-Vβ或Vβ-Cβ-L-Vα。
多篇文章描述了含天然二硫桥连接各亚基的TCR异二聚体的生产。尽管这些TCR可以被TCR-特异性抗体识别,但除非在相当高的浓度下,这些TCR无一显示能识别其天然配体,和/或它们不稳定。
WO 03/020763描述了可溶的TCR,该TCR折叠正确因此能识别其天然配体,能稳定一段时间,并能以合理的量制备。该TCR包含分别与TCRβ链胞外结构域二聚体化的TCRα链胞外结构域,通过引入各链恒定区内半胱氨酸之间的链间二硫键的方式实现二聚体化。
有人提出将特异性pMHC结合伴侣,即pMHC表位的特异性抗体,和二聚体与单链型的TCR作为靶向载体以便将治疗剂递送给抗原呈递细胞。为此目的,要求所述治疗剂与所述pMHC-结合伴侣以某种方式结合。建议与pMHC-结合伴侣一起用于此类靶向递送的治疗剂包括抗体(参见例如:Mosquera LA.等,J Immunol(2005)174(7):4381-8)、细胞因子(参见例如:Belmont HJ.等,Clin Immunol(2006)121(1):29-39;Wen J.等,CancerImmunol Immunother(2008)57(12):1781-94)和细胞毒剂。所述治疗剂是多肽时,与所述pMHC-结合伴侣结合的方式可以是通过肽融合与pMHC结合伴侣直接融合或经接头序列融合。在这些情况下,基本上只有两种融合可能性。单链抗体或TCR的情况下,所述融合原则上可以在TCR链的C-或N-末端;异二聚体抗体或TCR的情况下,所述融合原则上可以在各链的C-或N-末端。实际上,似乎所有pMHC结合伴侣-治疗剂融合子的已知示例都将治疗剂融合在C-末端(参见例如:Mosquera LA.等,J Immunol(2005)174(7):4381-8)、Belmont HJ.等,Clin Immunol(2006)121(1):29-39;Wen J.等,Cancer Immunol Immunother(2008)57(12):1781-94)。这是由于无论是单链还是异二聚体,抗体或TCR的功能取决于可变区的正确折叠与取向。治疗剂与pMHC结合伴侣的N-末端融合使治疗剂位于一个可变区前,本领域的设想认为位于N-末端的治疗剂将干扰抗体或TCR与pMHC复合体的结合,从而降低其结合效率。
发明内容
与本领域的设想相反,就诱导相关免疫应答而言,现发现免疫效应子部分融合于pMHC-结合伴侣N-末端的双功能分子,比所述融合位于pMHC-结合伴侣的C-末端的相应构建体更为有效。尽管所述N-和C-融合形式的pMHC结合亲和性相似,所述N-融合构建体的免疫应答增强。
发明详述
因此,本发明提供双功能分子,其包含特定pMHC表位的特异性多肽结合伴侣,以及免疫效应多肽,所述pMHC结合伴侣的N-末端与所述免疫效应多肽的C-末端连接,限制条件是所述多肽结合伴侣不是含SEQ ID No:7所示α链和SEQ ID No:9所示β链的T细胞受体。
如上所述,多肽pMHC结合伴侣可以是抗体或TCR。因此,本发明的一种实施方式中,所述pMHC结合伴侣是异二聚体αβ TCR多肽对,或单链αβ TCR多肽,所述异二聚体TCR多肽对的α或β链的N-末端或scTCR多肽的N-末端与所述免疫效应多肽的C-末端氨基酸连接。
pMHC结合伴侣与免疫效应多肽的连接可以是直接连接,或者是经接头序列的间接连接。接头序列通常是挠性的,由不含大体积侧链的氨基酸如甘氨酸、丙氨酸和丝氨酸组成,大体积侧链可能限制挠性。在任何特定pMHC结合伴侣-免疫效应子构建体的情况中,可容易地确定接头序列的可用或最优长度。接头序列通常为小于约12个氨基酸,例如小于10个氨基酸,或者5-10个氨基酸长。
在本发明的一些实施方式中,所述pMHC结合伴侣是异二聚体αβTCR多肽对,其中所述α和β多肽各自含有TCR可变区和恒定区,但没有TCR跨膜和胞质区。这种情况下,所述TCR部分是可溶的。在特别优选的此类双功能分子中,TCRα和β多肽恒定区残基之间存在非天然二硫键。具体地,α和β多肽的恒定区可通过取代TRAC 1的外显子1中Thr 48和TRBC1或TRBC2的外显子1中Ser 57的半胱氨酸之间的二硫键,或通过TRAC*01的外显子2中Cys4和TRBC1或TRBC2的外显子2中Cys2之间的天然二硫键连接。
在本发明的其它实施方式中,所述pMHC结合伴侣是Vα-L-Vβ、Vβ-L-Vα、Vα-Cα-L-Vβ或Vα-L-Vβ-Cβ型的单链αβTCR多肽,其中Vα和Vβ分别是TCRα和β可变区,Cα和Cβ分别是TCRα和β恒定区,L是接头序列。
免疫效应多肽是已知的。免疫效应多肽是通过直接或间接激活免疫系统的体液或细胞组分,例如通过T细胞激活,诱导或刺激免疫应答的分子。示例包括:IL-1、IL-1α、IL-3、IL-4、IL-5、IL-6、IL-7、IL-10、IL-11、IL-12、IL-13、IL-15、IL-21、IL-23、TGF-β、IFN-γ、TNFα、抗CD2抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体、抗CD44抗体、抗CD45RA抗体、抗CD45RB抗体、抗CD45RO抗体、抗CD49a抗体、抗CD49b抗体、抗CD49c抗体、抗CD49d抗体、抗CD49e抗体、抗CD49f抗体、抗CD16抗体、抗CD28抗体、抗IL-2R抗体、病毒蛋白和肽、和细菌蛋白或肽。所述免疫效应多肽是抗体时,所述抗体可用是scFv抗体,如抗CD3scFv。抗CD3抗体包括但不限于OKT3、UCHT-1、BMA031和12F6。
通过以下实施例说明本发明的原理。
实施例A.制备TCRβ链的C-或N-末端融合有效应多肽的可溶性αβTCR
A1.含抗CD3抗体作为效应多肽的可溶性NY-ESO TCR
本实施例的可溶性NY-ESO TCR能结合呈递在HLA-A2分子上的SLLMWITQV肽。
SEQ ID No:1(图1)是NY-ESO TCR的α链氨基酸序列,其中C162(采用SEQ ID No:1的编号)置换其TRAC恒定区的T48。
SEQ ID No:2(图2)是NY-ESO TCR的β链氨基酸序列,其中C170(采用SEQ ID No:2的编号)置换其TRBC2恒定区的S57。
SEQ ID No:3(图3)是抗CD3UCHT-1scFv抗体的氨基酸序列,其内部接头序列以下划线标出。
图4以框图形式显示可溶性NY-ESO αβTCR的结构,其含有SEQ IDNo:1所示α链和SEQ ID No:2所示β链,并含有SEQ ID No:3所示抗CD3UCHT-1 scFv抗体,所述抗体经接头序列L1融合在SEQ ID No:2所示TCRβ链的N-末端,L1为GGEGS(SEQ ID No:4)。
SEQ ID No:14(图16)为图2的β链氨基酸序列,其中抗CD3 scFv的N-末端经另一肽接头序列(下划线)融合到TCRβ链C-末端。
SEQ ID No:15(图17)为图2的β链氨基酸序列,其中抗CD3 scFv的C-末端经SEQ ID No 14中相同的肽接头序列(同样带下划线)融合到TCRβ链N-末端。
如下制备图4的构建体:
连接
将编码(a)TCRα链SEQ ID No:1和(b)SEQ ID No:2和SEQ ID No:3的融合序列的合成基因分别接入基于pGMT7的表达质粒,该质粒含有T7启动子以便在大肠杆菌BL21-DE3(pLysS)菌株中高水平表达(Pan等,Biotechniques(2000)29(6):1234-8)。
表达
所述表达质粒分别转化入大肠杆菌菌株BL21(DE3)Rosetta pLysS,氨苄青霉素抗性单克隆在TYP(氨苄青霉素100μg/ml)培养基中37℃生长至OD600为~0.6-0.8,然后用0.5mM IPTG诱导蛋白表达。诱导后3小时,在Beckman J-6B中以4000rpm离心30分钟收集细胞。用25ml Bug Buster(诺瓦基公司(Novagen)),在MgCl2和DNA酶存在下裂解细胞团块。在BeckmanJ2-21中以13000rpm离心30分钟回收包涵体团块。然后进行三次去污剂清洗以除去细胞碎片和膜组分。每次用曲通缓冲液(50mM Tris-HCI pH 8.0,0.5%Triton-X100,200mM NaCl,10mM NaEDTA)匀浆所述包涵体团块,然后在Beckman J2-21中以13000rpm离心15分钟。然后通过在以下缓冲液中的相似清洗除去去污剂和盐:50mM Tris-HCl pH 8.0,1mM NaEDTA。最后,将所述包涵体分成30mg的等份,-70℃冷冻。
重折叠
从冻存储备液中解冻约20mg的TCRα链和40mg的scFv-TCRβ链溶性包涵体,稀释成20ml胍溶液(6M盐酸胍,50mM Tris HCl pH 8.1,100mNaCl,10mM EDTA,20mM DTT),37℃水浴孵育30分钟到1小时确保链变性完全。然后,将含有完全还原和变性的TCR链的胍溶液注入1升下述折叠缓冲液中:100mM Tris pH 8.1,400mM L-精氨酸,2mM EDTA,5M尿素。在加入变性TCRα和scFv-TCRβ链前约5分钟,添加氧化还原对(盐酸半胱胺和二盐酸胱胺(终浓度分别为16mM和1.8mM))。所述溶液放置~30分钟。重折叠的scFv-TCR在渗析管纤维素膜(西格玛-艾尔德里奇公司(Sigma-Aldrich);产品编号D9402)中用10L H2O透析18-20小时。此后,用新鲜的10mM Tris pH 8.1(10L)更换透析缓冲液两次,在5℃±3℃下继续透析~8小时。通过下文所述3步纯化方法将可溶性且正确折叠的scFv-TCR与错误折叠物、降解产物和杂质分离。取决于所述可溶性抗CD3scFv-TCR融合体的pI,所述第二纯化步骤可以是离子交换层析或亲和层析。
第一纯化步骤
将透析重折叠物(10mM Tris pH8.1中)加载到POROS 50HQ阴离子交换柱上,用Akta纯化仪(GE医疗保健公司(GE Healthcare))以6个柱体积以上的0-500mM NaCl梯度洗脱结合的蛋白。峰组分(在电导率~20mS/cm下洗脱)在4℃保存。用经瞬时蓝染剂(Instant Blue Stain)(诺维克新公司(Novexin))染色的SDS-PAGE分析峰组分,然后合并。
第二纯化步骤
离子交换层析
阳离子交换纯化:
根据scFv-TCR融合体的pI,通过用20mM MES pH6-6.5稀释更换阴离子交换的合并组分的缓冲液。通过将稀释的合并组分(在20mM MESpH6-6.5中)加载到POROS 50HS阳离子交换柱上,使用Akta纯化仪(GE医疗保健公司)以6个柱体积以上的0-500mM NaCl梯度洗脱结合的蛋白,将可溶性且正确折叠的scFv-TCR与错误折叠物、降解产物和杂质分离。峰组分(在电导率~10mS/cm下洗脱)在4℃保存。
或者,可如下述使用采用羟基磷灰石基质的离子交换纯化。
羟基磷灰石层析:
通过用10mM NaH2PO4 pH6.0稀释更换阴离子交换合并组分的缓冲液。通过将稀释的合并组分(在10mM NaH2PO4 pH6.0中)加载到羟基磷灰石柱上,使用Akta纯化仪(GE医疗保健公司)以6个柱体积以上的10-500mMNaH2PO4/1M NaCl梯度洗脱结合的蛋白,将可溶性且正确折叠的scFv-TCR与错误折叠物、降解产物和杂质分离。峰组分(在电导率~20mS/cm下洗脱)在4℃保存。
亲和层析
对于pI接近6-6.5的一些scFv-TCR融合体,不能用离子交换步骤,但可以用亲和层析步骤替换。蛋白L亲和层析柱(皮尔斯公司(Pierce),产品编号89928)通过免疫球蛋白的κ轻链分离并纯化某些免疫球蛋白。蛋白L也能结合单链可变片段(scFv)。通过用PBS/0.02%叠氮钠稀释更换阴离子交换合并组分的缓冲液。通过将稀释的合并组分加载到蛋白L柱上,使用Akta纯化仪(GE医疗保健公司)以3个柱体积以上的0-25mM甘氨酸pH2.3/0.02%叠氮钠梯度洗脱结合的蛋白,将可溶性且正确折叠的scFv-TCR与错误折叠物、降解产物和杂质分离。scFv-TCR在所述梯度中很晚洗脱,通过添加TrispH8.1(100mM Tris pH8.1终浓度)中和洗脱组分的pH。峰组分在4℃下保存。
最后纯化步骤
用瞬时蓝染剂(诺维克新公司)染色的SDS-PAGE分析第二纯化步骤的峰组分,然后合并。然后为最后纯化步骤浓缩所述合并组分,在最后一步中用在PBS缓冲液(西格玛公司(Sigma))中预平衡的Superdex S200凝胶过滤柱(GE医疗保健公司)纯化并表征可溶性scFv-TCR。用瞬时蓝染剂(诺维克新公司)染色的SDS-PAGE分析相对分子量约78kDa时洗脱的峰,然后合并。
用制备图4的构建体相似的方式制备图5、6和7的构建体:
图5以框图形式显示可溶性NY-ESOαβTCR的结构,其含有SEQ IDNo:1所示α链和SEQ ID No:2所示β链,并含有SEQ ID No:3所示抗CD3UCHT-1scFv抗体,所述抗体经接头序列L2融合在SEQ ID No:2所示TCRβ链的N-末端,L2为AHHSEDPSSKAPKAP(SEQ ID No:5)。
图6以框图形式显示可溶性NY-ESO αβTCR的结构,其含有SEQ IDNo:1所示α链和SEQ ID No:2所示β链,并含有SEQ ID No:3所示抗CD3UCHT-1scFv抗体,所述抗体经接头序列L3融合在SEQ ID No:2所示TCRβ链的N-末端,L3为GGEGGGSEGGGS(SEQ ID No:6)。
图7以框图形式显示可溶性NY-ESO αβTCR的结构,其含有SEQ IDNo:1所示α链和SEQ ID No:2所示β链,并含有SEQ ID No:3所示抗CD3UCHT-1scFv抗体,所述抗体经接头序列L4融合在SEQ ID No:2所示TCRβ链的C-末端,在这一情况中L4为单个氨基酸S。
用针对图4、5、6和7的构建物所述的相似方式制备含SEQ ID No:1所示TCR α链和SEQ ID No:14所示TCR β链-抗CD3 scFv的融合蛋白,其中所述抗CD3 scFv融合在TCR β链的C-末端,或者含SEQ ID No:1所示TCR α链和SEQ ID No:15所示TCR β链-抗CD3 scFv的融合蛋白,其中所述抗CD3scFv融合在TCR β链的N-末端。
A2.含细胞因子作为效应多肽的可溶性嵌合型TCR
SEQ ID No:7(图8)是能结合小鼠胰岛素衍生肽LYLVCGERG(SEQ IDNO:8)的TCRα链的氨基酸序列,所述衍生肽由小鼠H-2Kd复合体呈递。(LYLVCGERG-H-2Kd),其中C158(采用SEQ ID No:7的编号)取代其TRAC恒定区的T48。
SEQ ID No:9(图9)是能结合小鼠LYLVCGERG-H-2Kd复合体的同一TCR的β链氨基酸序列,其中C171(采用SEQ ID No:9的编号)取代其TRBC2恒定区的S57。
SEQ ID No:7和9所示TCR是由α和βTCR链组成的嵌合型TCR,两条链各含有小鼠的可变区和人的恒定区。构建所述TCR的嵌合型形式以改善全小鼠TCR面临的重折叠问题,所述嵌合型TCR显示对小鼠胰岛素衍生肽-小鼠H-2Kd复合体的亲和性与小鼠TCR相同。
SEQ ID No:10(图10)是小鼠IL-4多肽的氨基酸序列。
SEQ ID No:11(图11)是小鼠IL-13多肽的氨基酸序列。
图12以框图形式显示可溶性嵌合型胰岛素αβTCR的结构,其含有SEQID No:7所示α链和SEQ ID No:9所示β链,并含有SEQ ID No:10所示小鼠IL-4,所述IL-4经接头序列L5融合在SEQ ID No:9所示TCRβ链的N-末端,L5为GGEGGGP(SEQ ID No:12)。
图13以框图形式显示可溶性嵌合型胰岛素αβTCR的结构,其含有SEQID No:7所示α链和SEQ ID No:9所示β链,并含有SEQ ID No:10所示小鼠IL-4,所述IL-4经接头序列L6融合在SEQ ID No:9所示TCRβ链的C-末端,L6为GSGGP(SEQ ID No:13)。
图14以框图形式显示可溶性嵌合型胰岛素αβTCR的结构,其含有SEQID No:7所示α链和SEQ ID No:9所示β链,并含有SEQ ID No:11所示小鼠IL-13,所述IL-13经接头序列L5融合在SEQ ID No:9所示TCRβ链的N-末端,L5为GGEGGGP(SEQ ID No:12)。
图15以框图形式显示可溶性嵌合型胰岛素αβTCR的结构,其含有SEQID No:7所示α链和SEQ ID No:9所示β链,并含有SEQ ID No:11所示小鼠IL-13,所述IL-13经接头序列L6融合在SEQ ID No:9所示TCRβ链的C-末端,L6为GSGGP(SEQ ID No:13)。
如下制备图12-15的构建体。
连接
将编码(a)TCRα链SEQ ID No:7和(b)SEQ ID No:9和SEQ ID No:10或11的融合序列的合成基因分别接入基于pGMT7的表达质粒,该质粒含有T7启动子以便在大肠杆菌BL21-DE3(pLysS)菌株中高水平表达(Pan等,Biotechniques(2000)29(6):1234-8)。
表达
所述含TCRα链和细胞因子β链的表达质粒分别转化入大肠杆菌菌株BL21(DE3)Rosetta pLysS,氨苄青霉素抗性单克隆在TYP(氨苄青霉素100μg/ml)培养基中37℃生长至OD600为~0.6-0.8,然后用0.5mM IPTG诱导蛋白表达。诱导后3小时,在Beckman J-6B中以4000rpm离心30分钟收集细胞。用25ml Bug Buster(诺瓦基公司),在MgCl2和DNA酶存在下裂解细胞团块。在Beckman J2-21中以13000rpm离心30分钟回收包涵体团块。然后进行三次去污剂清洗以除去细胞碎片和膜组分。每次用曲通缓冲液(50mM Tris-HCI pH 8.0,0.5%Triton-X100,200mM NaCl,10mM NaEDTA)匀浆所述包涵体团块,然后在Beckman J2-21中以13000rpm离心15分钟。然后通过在以下缓冲液中的相似清洗除去去污剂和盐:50mM Tris-HCl pH8.0,1mM NaEDTA。最后,将所述包涵体分成30mg的等份,-70℃冷冻。通过用6M盐酸胍溶解定量测定包涵体蛋白得率,利用日立(Hitachi)U-2001分光光度计获得OD测量值。然后采用理论消光系数计算蛋白浓度。
重折叠
从冻存储备液中解冻约20mg的TCRα链和40mg的细胞因子-TCRβ链溶性包涵体,稀释成20ml胍溶液(6M盐酸胍,50mM Tris HCl pH 8.1,100m NaCl,10mM EDTA,10mM DTT),37℃水浴孵育30分钟到1小时确保链变性完全。然后,将含有完全还原和变性的TCR链的胍溶液注入1升冷(5-10℃)的重折叠缓冲液中:100mM Tris pH 8.1,400mM L-精氨酸,2mM EDTA,5M尿素。在加入变性TCRα和细胞因子-TCRβ链前约5分钟,添加氧化还原对(盐酸半胱胺和二盐酸胱胺(终浓度分别为10mM和2.5mM))。所述溶液放置~30分钟。重折叠的细胞因子-TCR在渗析管纤维素膜(西格玛-艾尔德里奇公司;产品编号D9402)中用10L H2O透析18-20小时。此后,用新鲜的10mM Tris pH 8.1(10L)更换透析缓冲液两次,在5℃±3℃下继续透析~8小时。
纯化
通过下文所述3步纯化方法在室温下将可溶性细胞因子-TCR融合体与降解产物和杂质分离。
第一纯化步骤
在柱纯化前,用Sartopore 0.2μm滤囊(萨托瑞斯公司(Sartorius))过滤经透析的重折叠物。将过滤后的重折叠物加载到POROS 50HQ阴离子交换柱上,用Akta纯化仪(GE医疗保健公司)以6个柱体积以上的0-500mM NaCl梯度洗脱结合的蛋白。250mM NaCl下洗脱的峰组分含正确折叠的蛋白,将其在4℃保存。用经瞬时蓝染剂(Instant Blue Stain)(诺维克新公司)染色的SDS-PAGE分析峰组分,然后合并。
第二纯化步骤
混合含可溶性细胞因子-TCR的合并组分与等体积50mM Tris/1M(NH4)2SO4 pH 8,得到终浓度为0.5M(NH4)2SO4且室温下电导率为75-80mS/cm。通过将该样品加载到预平衡(50mM Tris/0.5M(NH4)2SO4pH 8)的丁基疏水相互作用柱(5ml HiTrap,GE医疗保健公司)上并用Akta纯化仪(GE医疗保健公司)收集流出相,将可溶性细胞因子-TCR与降解产物和杂质分离。含可溶性细胞因子-TCR的流出相样品用瞬时蓝染剂(诺维克新公司)染色的SDS-PAGE分析,然后合并,4℃保存。
最后纯化步骤
合并的组分用等体积10mM Tris pH8稀释,再浓缩至10ml(浓度≤3mg/ml)。用经PBS缓冲液(西格玛公司)预平衡的Superdex S200凝胶过滤柱(GE医疗保健公司)纯化可溶性细胞因子-TCR。用瞬时蓝染剂(诺维克新公司)染色的SDS-PAGE分析相对分子量约63kDa时洗脱的峰,然后合并。
实施例B.TCR β链C-或N-末端融合有效应多肽的可溶性αβTCR的性质
B1.含抗CD3抗体作为效应多肽的可溶性NY-ESO TCR
a.用与针对NY-ESO肽呈递细胞的抗CD3抗体融合的可溶性NY-ESOTCR重定向并激活CD8+T细胞
进行以下试验以证明抗CD3scFv-TCR融合体经特异性肽-MHC复合体激活细胞毒性T淋巴细胞(CTL)。采用ELISPOT试验测定的IFN-γ产量作为细胞毒性T淋巴细胞(CTL)激活的读取值和评估融合体中抗CD3 scFv部分的功效。
试剂
试验培养基:10%FCS(吉布可公司(Gibco),目录号2011-09),88%RPMI 1640(吉布可公司,目录号42401),1%谷氨酰胺(吉布可公司,目录号25030)和1%青霉素/链霉素(吉布可公司,目录号15070-063)。
肽:(SLLMWITQV)初始以4mg/ml溶解于DMSO(西格玛公司,目录号D2650)并冷冻。用所述肽脉冲处理T2细胞并用作靶细胞。
清洗缓冲液:0.01M PBS/0.05%吐温20
PBS(吉布可公司,目录号10010)
人IFNγELISPOT PVDF-酶促试剂盒(戴克隆公司,法国;目录号856.051.020)含有所有其它所需试剂。(捕获和检测抗体,脱脂奶粉,BSA,链霉亲和素-碱性磷酸酶和BCIP/NBT溶液,以及人IFN-γPVDF ELISPOT96孔板)
方法
靶细胞制备
用于此方法的靶细胞是(1)天然表位呈递细胞(例如Mel624或Mel526细胞),或是(2)如试剂部分所述经感兴趣的肽脉冲处理的T2细胞。通过在Megafuge 1.0(海芮斯公司(Heraeus))中以1200rpm离心3次清洗足量靶细胞(50000细胞/孔)。然后,细胞以106细胞/ml重悬于试验培养基。
效应细胞制备
本方法中所用效应细胞(T细胞)是CD8+T细胞(通过从PBL中阴性选择(采用CD8阴性分离试剂盒,戴纳尔公司(Dynal),目录号113.19)得到)、EBV细胞系的T细胞或PBMC。解冻效应细胞并放入试验培养基中,然后通过在Megafuge 1.0(海芮斯公司)中以1200rpm离心清洗细胞。然后,细胞以4X所需终浓度重悬于试验培养基。
试剂/测试化合物制备
通过稀释到试验培养基中制备不同浓度的测试化合物(所述TCR-抗CD3融合体;从10nM到0.03pM),得到4X终浓度。
ELISPOT
如下准备平板:每平板用10ml无菌PBS稀释100μl抗IFN-γ捕捉抗体。然后,每孔等分加入100μl稀释的捕捉抗体。该平板在4℃孵育过夜。孵育后,清洗平板(程序1,平板类型2,Ultrawash Plus 96孔板洗涤机;戴纳科斯公司(Dynex))以除去捕捉抗体。然后通过向各孔添加100μl含2%脱脂奶的无菌PBS并将平板在室温下孵育2小时进行平板封闭。然后,从板上洗去脱脂奶(程序1,平板类型2,Ultrawash Plus 96孔板洗涤机,戴纳科斯公司),通过在纸巾上轻弹和轻拍ELISPOT平板除去任何残留的清洗缓冲液。
然后,按以下顺序加入ELISPOT平板的试验组分:
50μl靶细胞,106细胞/ml(提供总计50000靶细胞/孔)
50μl试剂(所述抗CD3scFv-TCR融合体;不同浓度)
50μl培养基(试验培养基)
50μl效应细胞(1000-50000 CD8+细胞/孔;500-1000EBV细胞/孔;1000-50000PBMC/孔)。
然后,该平板孵育过夜(37℃/5%CO2)。次日,用清洗缓冲液清洗平板三次(程序1,平板类型2,Ultrawash Plus 96孔板洗涤机,戴纳科斯公司),在纸巾上轻拍除去过量清洗缓冲液。然后将100μL第一检测抗体加入各孔。所述第一检测抗体通过向戴克隆试剂盒中提供的一管检测抗体中添加550μl蒸馏水制备。然后将100μl该溶液稀释到10ml PBS/1%BSA(一块平板所需体积)中。然后,平板在室温下孵育至少2小时,然后用清洗缓冲液清洗3遍(程序1,平板类型2,Ultrawash Plus 96孔板洗涤机,戴纳科斯公司),通过将平板在纸巾上轻拍去除过量清洗缓冲液。
通过向各孔添加100μl稀释的链霉亲和素-碱性磷酸酶并将平板在室温下孵育1小时进行第二检测。所述链霉亲和素-碱性磷酸酶通过将10μl链霉亲和素-碱性磷酸酶溶液加入到10ml PBS/1%BSA(一块平板所需体积)中制得。然后用清洗缓冲液清洗平板三次(程序1,平板类型2,Ultrawash Plus96孔板洗涤机,戴纳科斯公司),在纸巾上轻拍除去过量清洗缓冲液。然后向各孔中加入100μl BCIP/NBT溶液,该溶液随戴克隆试剂盒提供。显影过程中,用铝箔封盖平板并放置5-15分钟。在此期间定时检查显影平板的斑点以确定终止反应的最优时间。然后用充满自来水的水槽清洗平板以终止显影反应,并振干平板,然后将其分解成3个组件。然后,平板在50℃干燥1小时,再用免疫斑点读数仪(Immunospot Plate reader)(CTL;细胞技术有限公司(Cellular Technology Limited))计数膜上形成的斑点。
结果
用ELISPOT试验(如上所述)测试了图4-7的抗CD3 scFv-TCR融合构建体。用Prism(GP公司(Graph Pad))将各孔观察到的ELISPOT斑点数对测试构建体的浓度作图。根据这些剂量-响应曲线,确定EC50值(EC50确定为引起最大响应的50%出的抗CD3scFv-TCR融合体浓度)。
测试构建体 | EC50 | EC50 | EC50 |
图7C-末端融合 | 5.044e-9 | 1.864e-9 | 2.383e-9 |
图5N-末端融合 | 8.502e-11 | ||
图4N-末端融合 | 4.825e-11 | ||
图6N-末端融合 | 3.95e-11 |
这些结果显示,图4、5和6的N-融合构建体激活细胞毒性T淋巴细胞的能力是图7的C-融合构建体的至少两倍。
b.用与抗CD3抗体融合的可溶性NY-ESO TCR重定向CD8+T细胞以杀伤IM9EBV转化B细胞系(非放射性细胞毒性试验)
进行下列试验以证明TCR-抗CD3 scFv融合体经特异性肽-MHC复合体激活细胞毒性T淋巴细胞(CTL),并评估所述融合体中抗CD3 scFv部分激活CTL杀死IM9细胞的效力。本试验是51Cr释放细胞毒性试验的比色替代试验,定量测定细胞裂解时释放的乳酸脱氢酶(LDH)。利用30分钟偶联的酶学实验测定培养物上清液中释放的LDH,该实验导致四唑盐(INT)转化为红色甲产物。形成的颜色深浅与裂解细胞数量成正比。用标准的96孔板读板仪在490nm下采集吸光数据。
材料
-CytoTox96非放射性细胞毒性试验(普洛麦格公司(Promega))(G1780)含底物混合物、试验缓冲液、裂解溶液和终止溶液
-试验培养基:10%FCS(热灭活,吉布可公司,目录号10108-165),88%RPMI 1640无酚红(英杰公司(Invitrogen),目录号32404014),1%谷氨酰胺,200mM(英杰公司,目录号25030024),1%青霉素/链霉素(英杰公司,目录号15070063)
-Nunc圆底微孔组织培养96孔板(纽恩克公司(Nunc),目录号163320)
-Nunc免疫学平板Maxisorb(纽恩克公司,目录号442404)
方法
靶细胞制备
本试验所用靶细胞是得自多发性骨髓瘤患者的IM9 EBV转化B细胞系(HLA-A2+NY-ESO+)。用Mel526黑色素瘤细胞系-HLA-A2+NY-ESO--作为对照。在试验培养基中制备靶细胞:靶细胞浓度调整为2x 105细胞/ml以在50μl中提供1x 104细胞/孔。
效应细胞制备
本试验所用效应细胞是CD8+T细胞。所用效应细胞对靶细胞的比例为10∶1(2x 106细胞/ml以在50μl中提供1x 105细胞/孔)。
试剂/测试化合物制备
如实施例A1中所述制备不同浓度的NY-ESO TCR-抗CD3融合体,其具有SEQ ID No:1所示TCR α链和SEQ ID No:14所示TCR β链-抗CD3scFv融合体,或者具有SEQ ID No:1所示TCRα链和SEQ ID No:15所示TCR β链-抗CD3 scFv融合体,并经本试验用试验培养基稀释备用(终浓度10-13-10-8M)。
试验准备
按以下顺序将试验组分加入平板:
-各孔中加入50μl靶细胞,IM9或Mel526(如前文所述制备)
-各孔中加入50μl试剂(如前文所述制备)
-各孔中加入50μl效应细胞(如前文所述制备)
如下所述制备多种对照:
-效应物自发释放:仅50μl效应细胞。
-靶细胞自发释放:仅50μl靶细胞。
-靶最大释放:50μl靶细胞,试验开始时添加80ug/ml地高辛以裂解细胞。
-试验培养基对照:仅150μl培养基。
实验孔一式三份制备,对照孔一式两份制备,终体积均为150μl。
平板以250x g离心4分钟,然后37℃下孵育24小时。
平板以250x g离心4分钟。从试验平板的各孔取37.5μl上清液移入平底96孔Nunc Maxisorb平板的对应孔。用试验缓冲液(12ml)重建底物混合物。向平板各孔加入37.5μl重建的底物混合物。平板用铝箔封盖,室温孵育30分钟。向平板各孔内加入37.5μl终止溶液以终止反应。添加终止溶液后1小时内在ELISA读板仪上记录490nm的吸光。
结果计算
从实验、靶细胞自发释放与效应细胞自发释放和靶最大释放的所有吸光值中减去培养基背景的平均吸光值。
初始两步所得的校正值用于下式以计算细胞毒性百分数:
%细胞毒性=100x(实验值-效应细胞自发值-靶细胞自发值)/(靶细胞最大值-靶细胞自发值)。
结果
用LDH释放试验(如上所述)测试含(i)TCRα链SEQ ID No:1和TCRβ链-抗CD3scFv融合体SEQ ID No:14(C-末端融合体)或(ii)TCRα链SEQID No:1和TCRβ链-抗CD3scFv融合体SEQ ID No:15(N-末端融合体)的NY-ESO TCR-抗CD3scFv融合体。用Prism(GP公司)将各孔观察到的%细胞毒性对测试构建体的浓度作图。根据这些剂量-响应曲线,确定EC50值(EC50确定为引起最大响应50%的TCR融合体浓度)。
这些结果显示,与含TCRα链SEQ ID No:1和TCRβ链-抗CD3 scFv融合体SEQ ID No:14的C-末端融合体相比,含TCRα链SEQ ID No:1和TCRβ链-抗CD3 scFv融合体SEQ ID No:15的N-末端融合体重定向细胞毒性T淋巴细胞以杀伤靶细胞的能力至少有效两倍。
B2.含细胞因子作为效应多肽的可溶性嵌合型TCR
b.小鼠IL-4细胞因子作为效应多肽
用下列试验测试图12-13的小鼠IL-4-TCR融合构建体中细胞因子部分的生物活性。该生物试验使用小鼠细胞系CTLL-2,该细胞的生长依赖小鼠IL-4,此处用于证明小鼠IL-4-TCR融合体中细胞因子部分的生物活性。
材料
CTLL-2细胞,普洛麦格CellTiter-Glo发光细胞活力试验(目录号G7572)包含CellTiter-Glo缓冲液和CellTiter-Glo底物(冻干)
试验培养基:含10%热灭活胎牛血清(吉布可公司,目录号10108-165),88%RPMI 1640(吉布可公司,目录号42401-018),1%谷氨酰胺(吉布可公司,目录号25030-024)和1%青霉素/链霉素(吉布可公司,目录号15070-063)的RPMI。
富集CTLL-2细胞,在试验培养基中清洗一次(1200rpm离心5分钟),计数并用台盼蓝溶液评估活力。若活力低于80%,进行菲可梯度离心以除去死细胞(无制动800xg离心15分钟)。细胞再清洗2次,调整体积使终浓度为1x 105细胞/ml。将CTLL-2细胞加入Nunc白色平底96孔板(5000细胞/孔),然后加入50μl已滴定浓度的标准小鼠IL-4(派普罗泰克公司(Peprotech))或图12与13的小鼠IL-4-嵌合型TCR融合构建体(7个点,1∶10稀释,从10-8到10-14M)。对照包括仅有细胞、仅有试验培养基、和细胞加200U/ml原白介素(凯隆公司(Chiron))。平板在37℃,5%CO2下孵育过夜。根据厂商指导,将CellTiter-Glo试剂解冻并加入平板(每孔100μl)。平板孵育10分钟以稳定发光信号,然后用发光读板仪记录信号。从读数中消去背景信号(仅有细胞),用Prism(GP公司)作图,以比较图12和13的小鼠IL-4-TCR融合构建体和“游离”重组小鼠IL-4的EC50。
结果
测试构建体 | EC50 | EC50 | EC50 |
m-IL4 | 4.984e-13 | 3.767e-13 | 5.148e-13 |
图13C-末端融合体 | 7.464e-12 | ||
图12N-末端融合体 | 5.913e-13 | 8.897e-13 |
这些结果显示,图12的N-融合构建体激活细胞毒性T淋巴细胞的能力是图13的C-融合构建体的至少两倍。
b.小鼠IL-13细胞因子作为效应多肽
用下列试验测试图14-15的小鼠IL-13-TCR融合构建体的细胞因子部分的生物活性。
进行该试验以证明细胞因子-TCR融合体中细胞因子部分的活性,即抑制人单核细胞产生IL-1β。该试验可用于测试细胞因子-TCR融合体,其中所述细胞因子是小鼠IL-13。
材料
衍生自暗黄层的单核细胞(暗黄层来自NBS BTS公司(NBS BristolTransfusion Service))。
戴纳尔公司用于原样人细胞的Dynabeads MyPure单核细胞试剂盒2(113.35)
试验培养基:10%热灭活胎牛血清(吉布可公司,目录号10108-165),88%RPMI 1640(吉布可公司,目录号42401-018),1%谷氨酰胺(吉布可公司,目录号25030-024)和1%青霉素/链霉素(吉布可公司,青霉素15070-063)
清洗缓冲液:0.01M PBS/0.05%吐温20(1袋含吐温20的磷酸缓冲盐水,pH7.4,西格玛公司,目录号P-3563,溶于1升蒸馏水,提供最终的组合物为0.01M PBS,0.138M NaCl,0.0027M KCl,0.05%吐温20)。
PBS(吉布可公司,目录号10010-015)。
无Ca2+与Mg2+的HBSS(吉布可公司,目录号1018-165)
细胞因子Eli-pair ELISA试剂盒:IL-1β(戴克隆公司,目录号DC-851.610.020),这些试剂盒含有所需的所有其它试剂,即捕捉抗体、检查生物素化抗体、链霉亲和素-HRP、IL-1β标准品、即用型TMB。以下方法基于随各试剂盒提供的使用说明书。
Nunc免疫学平板Maxisorb(纽恩克公司,目录号442404)。
Nunc圆底微孔组织培养96孔板(纽恩克公司,目录号163320)
BSA(西格玛公司,目录号A3059)
H2SO4(西格玛公司,目录号S1526)
台盼蓝(西格玛公司,目录号T8154)
源自大肠杆菌0111:B4的脂多糖(LPS)(西格玛公司,目录号L4391)
测试IL-13-TCR融合体试剂时所用的重组小鼠IL-13(派普罗泰克,目录号210-13)标准品。
单核细胞分离
从暗黄层分离PBMC:将暗黄层用HBSS(不含Ca2+与Mg2+)1∶2稀释,稀释后的血液层叠到淋巴细胞分离剂上(最多层叠35ml血液在15ml淋巴细胞分离剂上)以800xg(室温)无制动离心15分钟;取界面的细胞,用HBSS清洗并以1200rpm离心10分钟共4次。最后一次清洗后,细胞重悬于50ml试验培养基中,计数并用台盼蓝溶液评估活力。
用戴纳尔公司的Dynabeads MyPure单核细胞试剂盒2分离单核细胞。PBMC重悬于PBS/0.1%BSA,每107细胞加入100μl缓冲液、20μl封闭试剂和20μl抗体混合物,细胞在4℃孵育20分钟。清洗细胞并将重悬成每107细胞0.9ml PBS/0.1%BSA。加入预洗的珠(每107细胞100μl),混合并在20℃温和旋转再孵育15分钟。小心抽吸将花结细胞重悬,每107细胞加入1ml PBS/0.1%BSA。试管放入Dynal磁体2分钟。含负分离细胞的上清液移入新鲜试管并计数。立即使用细胞或将细胞冷冻在90%FCS/10%DMSO中备用。
细胞试验准备
ELISA平板用100μl/孔的PBS配制IL-1β捕捉抗体包被并4℃放置过夜。解冻单核细胞,在试验培养基中清洗2次并以5x 105细胞/毫升重悬。将单核细胞接种入圆底96孔板(每孔100μl,即每孔5x 104细胞)。通过用试验培养基稀释制备LPS、派普罗泰克重组细胞因子和测试细胞因子-TCR融合蛋白提供4X终浓度。向每孔内添加LPS(终浓度10ng/ml),然后按三复孔添加50μl滴定浓度(6个点,1∶10连续稀释)的派普罗泰克重组IL-13(终浓度10-8-10-13M)或测试细胞因子-TCR融合蛋白(终浓度10-7-10-13M)。平板在37℃,5%CO2下孵育过夜。
IL-1β ELISA
抗体包被的IL-1β ELISA平板用清洗缓冲液清洗3次,然后用250μlPBS/5%BSA/孔在室温下封闭至少2小时(或者4℃过夜)。ELISA平板用清洗缓冲液清洗3次并拍干。IL-1β标准品用PBS/1%BSA稀释。含细胞的平板以1200rpm离心5分钟。然后将各孔的上清液转移至预包被的IL-1βELISA平板。将100μl细胞上清液(用PBS/1%BSA 1∶3稀释)或标准品加入相应孔内,并每孔加入50μl检测抗体(按试剂盒的说明稀释)。平板在室温下培育2小时。用清洗缓冲液清洗3次平板。每孔加入100μl链霉亲和素-HRP(按试剂盒使用说明书稀释),将平板在室温下孵育20分钟。用清洗缓冲液清洗3次平板。每孔加入100μl即用TMB,使平板在暗处(铝箔下)显影5-20分钟(取决于信号强度)。通过添加100μl/孔的1M H2SO4终止反应。
在微板读数仪上读取平板在450nm的平板吸光,参比滤光片设为650nm。各滴定点的抑制量计算为产生最大信号的含单核细胞与LPS而没有细胞因子-TCR融合蛋白的样品的百分数,从而产生剂量-效应曲线。
结果
测试构建体 | EC50 | EC50 |
m-IL13 | 1.535e-10 | 9.534e-11 |
图15C-末端融合体 | 6.21e-10 | |
图14N-末端融合体 | 1.493e-10 |
这些结果显示,图14的N-融合构建体抑制人单核细胞产生IL-1β的能力是图15的C-融合构建体的至少两倍。
Claims (14)
1.一种双功能分子,其包含特定pMHC表位的特异性多肽结合伴侣,和免疫效应多肽,所述pMHC结合伴侣是异二聚体αβTCR多肽对或单链αβTCR多肽,所述异二聚体TCR多肽对的α或β链的N-末端或所述scTCR多肽的N-末端与所述免疫效应多肽的C-末端氨基酸连接,限制条件是所述多肽结合伴侣不是含SEQ ID No:7所示α链和SEQ ID No:9所示β链的T细胞受体。
2.如权利要求1所述的双功能分子,其特征在于,所述pMHC结合伴侣是异二聚体αβTCR多肽对,其中所述α和β多肽各自含有TCR可变区和恒定区,但没有TCR跨膜和胞质区。
3.如权利要求2所述的双功能分子,其特征在于,所述α和β多肽的恒定区通过取代TRAC1的外显子1中Thr48和TRBC1或TRBC2的外显子1中Ser57的半胱氨酸之间的二硫键,或通过TRAC1的外显子2中Cys4和TRBC1或TRBC2的外显子2中Cys2之间的天然二硫键连接。
4.如权利要求1所述的双功能分子,其特征在于,所述pMHC结合伴侣是单链αβTCR多肽。
5.如上述权利要求中任一项所述的双功能分子,其特征在于,所述免疫效应多肽是特异性结合T细胞所呈递抗原的抗体。
6.如权利要求5所述的双功能分子,其特征在于,所述抗体是scFv抗体。
7.如权利要求5所述的双功能分子,其特征在于,所述抗体是抗CD3抗体。
8.如权利要求6所述的双功能分子,其特征在于,所述抗体是抗CD3抗体。
9.如权利要求7所述的双功能分子,其特征在于,所述抗体是OKT3。
10.如权利要求7所述的双功能分子,其特征在于,所述抗体是UCHT-1。
11.如权利要求7所述的双功能分子,其特征在于,所述抗体是BMA031。
12.如权利要求7所述的双功能分子,其特征在于,所述抗体是12F6。
13.如权利要求1-4、6-12中任一项所述的双功能分子,其特征在于,其中的效应多肽是调节淋巴细胞免疫抑制或免疫刺激活性的细胞因子。
14.如权利要求5所述的双功能分子,其特征在于,其中的效应多肽是调节淋巴细胞免疫抑制或免疫刺激活性的细胞因子。
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