CN1921889A - 靶定的免疫原 - Google Patents
靶定的免疫原 Download PDFInfo
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- CN1921889A CN1921889A CNA2004800421537A CN200480042153A CN1921889A CN 1921889 A CN1921889 A CN 1921889A CN A2004800421537 A CNA2004800421537 A CN A2004800421537A CN 200480042153 A CN200480042153 A CN 200480042153A CN 1921889 A CN1921889 A CN 1921889A
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Abstract
本发明提供了用于产生和利用靶定的免疫原的试剂和方法。在优选实施方案中,将免疫原缀合到将该免疫原靶向MHC呈递途径的氨基酸序列。使用本文提供的试剂和方法,可以增强免疫方案,从而导致增加的宿主免疫性。
Description
相关申请
本申请要求2003年12月31日提交的美国临时申请号60/533,728的优先权。
发明领域
本发明涉及用于改进免疫方案的试剂和方法。例如,将免疫原性氨基酸序列导向MHC呈递途径的氨基酸序列。
发明背景
尽管基于肽的疫苗有许多优点(安全、易于生产),但是它们显示出有限的免疫原性。这部分是由于外源肽不能有效进入I类MHC呈递途径。从而,可以增强肽向MHC递送的策略具有增加基于肽的疫苗的功效的潜力。一种策略是将免疫原性序列连接到“蛋白质转导结构域”(PTD),已经表明所述结构域驱动蛋白质和肽越过细胞膜的转运。示例性PTD包括HIT-Tat、细胞穿透肽(CPP)、Trojan载体、触角足同源域和人period-1蛋白。
在一种方法中,抗原性肽附着到来自HIV-1 tat的短阳离子肽(即,残基49-57)以形成融合缀合物。已经表明抗原呈递细胞(“APC”),如树突细胞的暴露可加工ova-tat缀合物,从而导致抗原特异的CD8+T细胞的刺激(Kim,等人J Immunol 1997 Aug 15;159(4):1666-8;Shibagaki,等人J Immunol 2002 Mar 1;168(5):2393-401)。这也已经对人黑素瘤抗原TRP2得到了证明(Wang,等人J Clin Invest 2002 Jun;109(11):1463-70)。将tat肽缀合到全长蛋白质后证明了相反的证据(Leifert,等人Gene Ther2002 Nov;9(21):1422-8)。
在另一方法中,将触角足同源域(AntpHD)与CTL表位融合并且表明其增强了CD8+T细胞反应性(Chikh,等人J Immunol 2001Dec 1;167(11):6462-70;Pietersz,等人Vaccine 2001 Jan 8;19(11-12):1397-405;Schutze-Redelmeier,等人J Immunol 1996 Jul15;157(2):650-5)。已经表明可以使用具有最多达50个氨基酸的抗原性序列的AntpHD。
在其他研究中,已经表明来自人period-1蛋白质的转导序列(hPER1,序列SRRHHCRSKAKRSRHH)有效越过细胞膜。因此,它是吸引人的抗原递送载体候选物。如下面详细描述的,hPER1实际上增强抗原呈递和T细胞反应性。
附图简述
图1.通过hPER1缀合物体外敏化靶细胞进行肽特异裂解。
图2.用hPER1缀合物肽体外诱导人T细胞应答。
图3.用hPER1缀合物肽不用佐剂体内诱导T细胞应答。
图4.静脉内(i.v.)注射肽脉冲的DC后C57BL/6小鼠中的CTL应答。用经所指示的肽脉冲的5×105个骨髓来源的DC静脉内免疫小鼠。免疫后一周从接种的动物收获脾细胞,用SIINFEKL肽再刺激5天,并在标准铬释放测定中用SIINFEKL肽脉冲的靶细胞测试CTL活性。
图5.皮下(s.c.)注射肽后HLA-A2/Kb转基因小鼠中的CTL应答。将小鼠用50μg所指示的肽皮下免疫并在第一次注射后第21和42天加强免疫。在免疫后63天收获来自免疫动物的脾细胞,用天然gp100-154肽再刺激5天,并在标准铬释放测定中用gp100-154肽脉冲的靶细胞测试CTL活性。
图6.转基因A2/Kb小鼠中hPER1-FVYVW-154介导CTL应答可以通过不同的免疫途径产生。所示结果代表每组的四只个别小鼠的平均值。
图7.用缀合SIINFEKL表位的hPER1或者Tat肽体内诱导T细胞应答。用通过DEVWEL接头序列结合于Tat或者hPER1的SIINFEKL肽皮下免疫小鼠。该图中所示结果代表每组的四只个别小鼠的平均值。与IFA中的阳性对照SIINFEKL相比,hPER1-DEVWEL-SIINFEKL得到最佳的CTL应答。
图8.辅助CD4乙型肝炎肽的存在对于产生针对CD8肽的CTL应答是必需的。用从50纳摩尔到1纳摩尔的不同剂量的hPER1-FVYVW-154肽用或者不用辅助肽鼻内接种A2/Kb小鼠。不存在辅助肽时,10纳摩尔hPER1-FVYVW-154不诱导显著细胞毒性。
图9.不存在辅助肽时,用较高的肽剂量免疫可以在小鼠中诱导T细胞应答。用不同剂量的hPER1-SGQL-SIINFEKL用或者不用辅助肽皮内免疫C57BL/6小鼠。
图10.在不同接头序列存在下,用结合SIINFEKL的hPER1进行无佐剂的肽免疫后体内诱导免疫性。结果显示了每组的4只个别小鼠的平均值。FVYVW接头已经产生了最显著的CTL杀伤,其与存在不完全弗氏佐剂(IFA)时的SIINFEKL免疫相当。
图11.OVA(SIINFEKL)肽呈递的体外分析。将来自C57BL/6小鼠的脾细胞用10μg/ml所指示的肽在37℃脉冲1小时,洗涤,并温育0、4、8、24或30小时。使用转导肽脉冲的细胞与bGAL肽预温育以阻断任何细胞表面结合。然后通过ELISPOT测试细胞诱导从SIINFEKL特异T细胞分泌IFN-γ的能力。大于300/孔的点数不能计数。*=未测试的样品。
图12.NP肽呈递的体外分析。将来自C57BL/6小鼠的脾细胞用10μg/ml所指示的肽在37℃脉冲1小时,洗涤,并温育0、24、72或120小时。通过ELISPOT测试细胞诱导从NP-特异T细胞分泌IFN-γ的能力。
图13.hPER1-FVYVW-gp100-154肽免疫后长期免疫性的诱导。仅用gp100-154肽或者结合hPER1-FVYVW皮下免疫3周或者3个月后A2/Kb小鼠中的CTL应答。结果显示了单个小鼠(4只小鼠/组)。hPEr1-FVYVW-154免疫后分别在4/4或者3/4小鼠中观察到短期(3周)以及长期(3个月)T细胞应答。相比而言,仅用154免疫不产生显著CTL应答。
发明概述
本发明提供了用于产生和利用靶定的免疫原的试剂和方法。在优选实施方案中,将免疫原缀合到氨基酸序列,所述序列将免疫原靶向MHC用于呈递。使用本文提供的试剂和方法,可以增强免疫方案,从而导致宿主的增强的免疫性。
详述
本发明提供了用优选将肽导向MHC呈递途径的氨基酸序列(本文中称作“靶向序列”)将免疫原靶向MHC途径的方法。该靶向策略可以用于例如基于肽的免疫方案中,以用于在树突细胞中表达抗原,用于核酸疫苗,和基于载体(即,病毒、细菌)的接种。为了描述本发明,将连接到靶向氨基酸序列的免疫原性氨基酸序列称作“靶定的免疫原”。术语“靶定的免疫原”包括其片段、变体或者衍生物。
靶向序列可以包括,例如,本领域已知的任意转导序列。优选来自触角足、TAT、VP22或hPER1蛋白的序列(即,靶向序列)。更优选的靶向序列包括例如:
TAT:GYGRKKRRQRRR(SEQ ID NO.:1)
AntP:RQIKIWFQNRRMKWKK(SEQ ID NO.:2)
PER1-1:SRRHHCRSKAKRSRHH(SEQ ID NO.:3)
PER1-2:RRHHRRSKAKRSR(SEQ ID NO.:4)
在一个实施方案中,细胞毒性T淋巴细胞(CTL)表位结合到hPER1转导序列以形成靶定的免疫原(或者“hPER1-CTL缀合物”)。优选对宿主施用靶定的免疫原导致抗免疫原免疫应答,其大于仅用该免疫原得到的免疫应答(即,增加的细胞毒性T细胞应答)。
适宜的免疫原还可以包括例如,肿瘤抗原(TA)的肽序列。术语“TA”包括肿瘤相关抗原(TAA)和肿瘤特异抗原(TSA),其中癌性细胞是抗原的来源。TAA是在肿瘤细胞表面上以高于正常细胞上观察到的量表达的抗原或者在胎儿发育期间在正常细胞上表达的抗原。TSA是肿瘤细胞独特的抗原并且不在正常细胞上表达。TA还包括TAA或者TSA、其抗原性或者免疫原性片段,和保留它们的抗原性和/或免疫原性的修饰形式。通常将TA根据它们的表达模式、功能或者遗传来源分成5类:癌-睾丸(CT)抗原(即,MAGE,NY-ESO-1);黑素细胞分化抗原(即,Melan A/MART-1,酪氨酸酶,gp100);突变抗原(即,MUM-1,p53,CDK-4);超表达的‘自身’抗原(即,HER-2/neu,p53);和病毒抗原(即,HPV,EBV)。适宜的TA包括例如,gp100(Cox等人,Science,264:716-719(1994))、MART-1/Melan A(Kawakami等人,J.Exp.Med.,180:347-352(1994))、gp75(TRP-1)(Wang等人,J.Exp.Med.,186:1131-1140(1996))、酪氨酸酶(Wolfel等人,Eur.J.Immunol.,24:759-764(1994))、NY-ESO-1(WO 98/14464;WO 99/18206)、黑素瘤蛋白聚糖(Hellstrom等人,J.Immunol.,130:1467-1472(1983))、MAGE家族抗原(即,MAGE-1、2、3、4、6和12;Van der Bruggen等人,Science,254:1643-1647(1991);美国专利号6,235,525)、BAGE家族抗原(Boel等人,Immunity,2:167-175(1995))、GAGE家族抗原(即,GAGE-1、2;Van der Eynde等人,J.Exp.Med.,182:689-698(1995);美国专利号6,013,765)、RAGE家族抗原(即,RAGE-1;Gaugler等人,Immunogenetics,44:323-330)(1996);美国专利号5,939,526)、N-乙酰葡糖胺转移酶-V(Guilloux等人,J.Exp.Med.,183:1173-1183(1996))、p15(Robbins等人,J.1mmunol.,154:5944-5950(1995))、β-连环蛋白(Robbins等人,J.Exp.Med.,183:1185-1192(1996))、MUM-1(Coulie等人,Proc.Natl.Acad.Sci.USA,92:7976-7980(1995))、依赖细胞周期蛋白的激酶-4(CDK4)(Wolfel等人,Science,269:1281-1284(1995))、p21-ras(Fossum等人,Int.J.Cancer,56:40-45(1994))、BCR-abl(Bocchia等人,Blood,85:2680-2684(1995))、p53(Theobald等人,Proc.Natl.Acad.Sci.USA,92:11993-11997(1995))、p185HER2/neu(erb-B1;Fisk等人,J.Exp.Med.,181:2109-2117(1995))、表皮生长因子受体(EGFR)(Harris等人,Breast Cancer Res.Treat,29:1-2(1994))、癌胚抗原(CEA)(Kwong等人,J.Natl.Cancer Inst.,85:982-990(1995)、美国专利号5,756,103;5,274,087;5,571,710;6,071,716;5,698,530;6,045,802;EP 263933;EP 346710;和EP 784483);癌相关的突变粘蛋白(即,MUC-1基因产物;Jerome等人,J.Immunol.,151:1654-1662(1993));EBV的EBNA基因产物(即,EBNA-1;Rickinson等人,Cancer Surveys,13:53-80(1992));人乳头瘤病毒的E7,E6蛋白质(Ressing等人,J.Immunol,154:5934-5943(1995));前列腺特异抗原(PSA;Xue等人,The Prostate,30:73-78(1997));前列腺特异膜抗原(PSMA;Israeli,等人,Cancer Res.,54:1807-1811(1994));独特型表位或者抗原,例如,免疫球蛋白独特型或者T细胞受体独特型(Chen等人,J.Immunol.,153:4775-4787(1994));KSA(美国专利号5,348,887)、驱动蛋白2(Dietz,等人Biochem Biophys Res Commun 2000 Sep 7;275(3):731-8)、HIP-55、TGFβ-1抗细胞凋亡因子(Toomey,等人Br J Biomed Sci 2001;58(3):177-83)、肿瘤蛋白D52(Bryne J.A.,等人,Genomics,35:523-532(1996))、H1FT、NY-BR-1(WO01/47959)、NY-BR-62、NY-BR-75、NY-BR-85、NY-BR-87和NY-BR-96(Scanlan,M.Serologic and Bioinformatic Approaches to theIdentification of Human Tumor Antigens,in Cancer Vaccines 2000,Cancer Research Institute,New York,NY),包括野生型、修饰的、突变的TA以及其免疫原性片段和衍生物。这些TA的任一种可以单独使用或者与一种或多种靶定的免疫原组合用于共免疫方案中。
许多适宜的TA来源的肽序列适于用于实践本发明。优选的TA来源的肽序列在下面显示,它们的任一种都可以连接靶向序列,如TAT、AntP、hPER1-1或者hPER1-2:
gp100-280-288(9V) YLEPGPVTV (SEQ ID NO:5)
gp100-154-162 KTWGQYWQV (SEQ ID NO:6)
MART-1 32 ILTVILGVL (SEQ.ID.NO.7)
MART-1 31 GILTVILGV (SEQ.ID.NO.8)
MART-1 99 NAPPAYEKL (SEQ.ID.NO.9)
MART-1 1 MPREDAHFI (SEQ.ID.NO.10)
MART-1 56 ALMDKSLHV (SEQ ID.NO.11)
MART-1 39 VLLLIGCWY (SEQ.ID.NO.12)
MART-1 35 VILGVLLLI (SEQ.ID.NO.13)
MART-1 61 SLHVGTQCA (SEQ.ID.NO.14)
MART-1 57 LMDKSLHVG (SEQ.ID.NO.15)
MAGE-A3 115 ELVHFLLLK (SEQ ID NO:16)
MAGE-A3 285 KVLHHMVKI (SEQ ID NO:17)
MAGE-A3 276 RALVETSYV (SEQ ID NO:18)
MAGE-A3 105 FQAALSRKV (SEQ ID NO:19)
MAGE-A3 296 GPHISYPPL (SEQ ID NO:20)
MAGE-A3 243 KKLLTQHFV (SEQ ID NO.21)
MAGE-A3 24 GLVGAQAPA (SEQ ID NO.22)
MAGE-A3 301 YPPLHEWVL (SEQ ID NO.23)
MAGE-A3 71 LPTTMNYPL (SEQ ID NO.24)
Tyr 171 NIYDLFVWM (SEQ ID NO:25)
Tyr 444 DLGYDYSYL (SEQ ID NO:26)
Tyr 57 NILLSNAPL (SEQ ID NO:27)
TRP-1 245 SLPYWNFAT (SEQ ID NO:28)
TRP-1 298 TLGTLCNST (SEQ ID NO:29)
TRP-1 481 IAVVGALLL (SEQ ID NO:30)
TRP-1 181 NISIYNYFV (SEQ ID NO:31)
TRP-1 439 NMVPFWPPV (SEQ ID NO:32)
额外适宜的免疫原包括来自感染性生物的那些免疫原,所述感染性生物包括细菌、病毒、寄生物等等。例如,百日咳抗原如百日咳毒素、丝状血凝素、百日咳杆菌粘附素(pertactin)、凝集原或者从它们衍生的肽与靶向序列如hPER1-1或者hPER1-2融合后可以用作疫苗。类似地,来自如本领域已知的除其他以外的致病生物如棒杆菌(Corynebacterium)(即,白喉棒杆菌)、梭菌(Clostridium)(即,破伤风梭菌)、奈瑟氏球菌(Neisseria)(即,脑膜炎奈瑟氏球菌)、链球菌(Streptococcus)、嗜血菌(Hemophilus)、脊髓灰质炎病毒、流感病毒、肝炎病毒、人免疫缺陷病毒(HIV)的抗原也可以使用。
在一些实施方案中,可以用插入靶向序列和免疫原序列之间的接头序列连接靶向序列和免疫原性肽序列。适宜的接头包括例如,在肽所来源的全长亲代多肽中天然存在于所述肽序列的N-末端的氨基酸序列。例如,在全长gp100多肽内gp100肽序列KTWGQYWQV天然地在其N末端具有序列FVYVW。因此,FVYVW可以用于连接gp100肽与靶向序列。其他适宜的接头可以用设计与MHC分子相互作用的肽的标准方法设计,如本领域已知的。
一些实施方案还可以包括本发明的肽序列的衍生物。一种类型的衍生物是其中一个氨基酸序列用另一个替代的序列。替代可以是保守的,或者非保守的,或者其任意组合。对多肽序列的保守氨基酸修饰(和对编码核苷酸的相应修饰)可以产生具有类似于亲代多肽的功能和化学特征的多肽。例如,“保守氨基酸替代”可以涉及将天然氨基酸残基用非天然残基替代从而对该位置的氨基酸残基的大小、极性、电荷、疏水性或者亲水性有很小的影响或者没有影响,并且,尤其不导致减小的免疫原性。适宜的保守氨基酸替代在表I中显示。
表I
原来的残基 | 示例性替代 | 优选的替代 |
Ala | Val,Leu,Ile | Val |
Arg | Lys,Gln,Asn | Lys |
Asn | Gln | Gln |
Asp | Glu | Glu |
Cys | Ser,Ala | Ser |
Gln | Asn | Asn |
Glu | Asp | Asp |
Gly | Pro,Ala | Ala |
His | Asn,Gln,Lys,Arg | Arg |
Ile | Leu,Val,Met,Ala,Phe,正亮氨酸 | Leu |
Leu | 正亮氨酸,Ile,Val,Met,Ala,Phe | Ile |
Lys | Arg,1,4二氨基-丁酸,Gln,Asn | Arg |
Met | Leu,Phe,Ile | Leu |
Phe | Leu,Val,Ile,Ala,Tyr | Leu |
Pro | Ala | Gly |
Ser | Thr,Ala,Cys | Thr |
Thr | Ser | Ser |
Trp | Tyr,Phe | Tyr |
Tyr | Trp,Phe,Thr,Ser | Phe |
Val | Ile,Met,Leu,Phe,Ala,正亮氨酸 | Leu |
技术人员将能够用公知的技术确定免疫原性靶的适宜的变体。为了鉴定分子中可以改变而不破坏生物活性(即,MHC结合、免疫原性)的区域,本领域技术人员可以靶向认为对于该活性不重要的区域。例如,当来自相同物种或者其他物种的具有相似活性的免疫原性靶是已知的时候,本领域技术人员可以比较多肽与此类相似的多肽的氨基酸序列。通过进行此类分析,人们可以鉴定保守的残基和分子部分。将明白相对于此类相似免疫原性靶不保守的分子区域中的改变将较不可能不利地影响多肽的生物活性和/或结构。本领域技术人员将还已知,甚至在相对保守的区域中,人们也可以用化学相似的氨基酸替代天然发生的残基而保留活性。因此,甚至对生物活性或者结构重要的区域也可以进行保守氨基酸替代而不破坏免疫原性靶的生物活性或者不会不利地影响它的结构。
在一些实施方案中,将编码所述肽序列的核酸分子插入到表达载体中,如下文更详细地讨论的。在此类实施方案中,所述肽序列由对应于氨基酸序列的核苷酸编码。编码多种氨基酸的核苷酸的特定组合是本领域公知的,如本领域技术人员使用的多种参考文献中描述的(即,Lewin,B.Genes V,Oxford University Press,1994),如下面的表II中所示:
表II
Phe | TTT | Ser | TCT | Tyr | TAT | Cys | TGT |
TTC | TCC | TAC | TGC | ||||
Leu | TTA | TCA | TERM | TAA | TERM | TGA | |
TTG | TCG | TAG | Trp | TGG | |||
CTT | Pro | CCT | His | CAT | Arg | CGT | |
CTC | CCC | CAC | CGC | ||||
CTA | CCA | Gln | CAA | CGA | |||
CTG | CCG | CAG | CGG | ||||
Ile | ATT | Thr | ACT | Asn | AAT | Ser | AGT |
ATC | ACC | AAC | AGC | ||||
ATA | ACA | Lys | AAA | Arg | AGA | ||
Met | ATG | ACG | AAG | AGG | |||
Val | GTT | Ala | GCT | Asp | GAT | Gly | GGT |
GTC | GCC | GAC | GGC | ||||
GTA | GCA | Glu | GAA | GGA | |||
GTG | GCG | GAG | GGG |
编码本发明的多种肽的示例性DNA序列在下面显示:
TAT (SEQ ID NO.:33):
GGCTACGGCAGGAAGAAGAGGAGGCAGAGGAGGAGG
AntP (SEQ ID NO.:34):
AGGCAGATCAAGATCTGGTTCCAGAACAGGAGGATGAAGTGGAAGAAG
PER1-1(SEQ ID NO.:35):
AGCAGGAGGCACCACTGCAGGAGCAAGGCCAAGAGGAGCAGGCACCAC
PER1-2(SEQ ID NO.:36):
AGGAGGCACCACAGGAGGAGCAAGGCCAAGAGGAGCAGG
gp100-280-288(9V):
TACCTGGAGCCCGGCCCCGTGACCGTG (SEQ ID NO.:37)
gp100-154-162:
AAGACCTGGGGCCAGTACTGGCAGGTG (SEQ ID NO.:38)
MART-1 32: ATCCTGACAGTGATCCTGGGAGTCTTA (SEQ ID NO:39)
MART-1 31: GGCATCCTGACAGTGATCCTGGGAGTC (SEQ ID NO:40)
MART-1 99: AATGCTCCACCTGCTTATGAGAAACTC (SEQ ID NO:42)
MART-1 1: ATGCCAAGAGAAGATGCTCACTTCATC (SEQ ID NO:43)
MART-1 56: GCCTTGATGGATAAAAGTCTTCATGTT (SEQ ID NO:44)
MART-1 39: GTCTTACTGCTCATCGGCTGTTGGTAT (SEQ ID NO:45)
MART-1 35: GTGATCCTGGGAGTCTTACTGCTCATC (SEQ ID NO:46)
MART-1 61: AGTCTTCATGTTGGCACTCAATGTGCC (SEQ IDNO:47)
MART-1 57: TTGATGGATAAAAGTCTTCATGTTGGC (SEQ ID NO:48)
MAGE-A3 115:GAGTTGGTTCATTTTCTGCTCCTCAAG (SEQ ID NO.49)
MAGE-A3 285:AAAGTCCTGCACCATATGGTAAAGATC (SEQ.ID.NO.50)
MAGE-A3 276:AGGGCCCTCGTTGAAACCAGCTATGTG (SEQ ID.NO.51)
MAGE-A3 105:TTCCAAGCAGCACTCAGTAGGAAGGTG (SEQ ID.NO.52)
MAGE-A3 296:GGACCTCACATTTCCTACCCACCCCTG (SEQ.ID.NO.53)
MAGE-A3 243:AAGAAGCTGCTCACCCAACATTTCGTG (SEQ ID.NO.54)
MAGE-A3 24: GGCCTGGTGGGTGCGCAGGCTCCTGCT (SEQ ID NO:55)
MAGE-A3 301:TACCCACCCCTGCATGAGTGGGTTTTG (SEQ ID.NO.56)
MAGE-A3 71: CTCCCCACTACCATGAACTACCCTCTC(SEQ.ID.NO.57)
TYR 171: AATATTTATGACCTCTTTGTCTGGATG (SEQ ID NO:58)
TYR 444: GATCTGGGCTATGACTATAGCTATCTA (SEQ ID NO:59)
TYR 57: AATATCCTTCTGTCCAATGCACCACTT (SEQ ID NO:60)
TRP-1 245: TCCCTTCCTTACTGGAATTTTGCAACG (SEQ ID NO:61)
TRP-1 298: ACCCTGGGAACACTTTGTAACAGCACC (SEQ ID NO:62)
TRP-1 481: ATAGCAGTAGTTGGCGCTTTGTTACTG (SEQ ID NO:63)
TRP-1 181: AACATTTCCATTTATAACTACTTTGTT (SEQ ID NO:64)
TRP-1 439: AACATGGTGCCATTCTGGCCCCCAGTC (SEQ ID NO:65)
下面显示了示例性免疫原性靶的氨基酸和DNA序列,所述免疫原性靶包括代表靶向序列的第一个氨基酸和代表免疫原(T细胞表位)的第二个氨基酸序列:
hPER1-1-gp100(280-288)
S R R H H C R S K A K R S R H
AGC AGG AGG CAC CAC TGC AGG AGC AAG GCC AAG AGG AGC AGG
CAC
H Y L E P G P V T V
CAC TAC CTG GAG CCC GGC CCC GTG ACC GTG (SEQ ID NO:66)
hPER1-2-gp100(154-162)
R R H H R R S K A K R S R
AGG AGG CAC CAC AGG AGG AGC AAG GCC AAG AGG AGC AGG
K T W G Q Y W Q V
AAG ACC TGG GGC CAG TAC TGG CAG GTG (SEQ ID NO:67)
hPER1-2-F-gp100(154-162)
R R H H R R S K A K R S R
AGG AGG CAC CAC AGG AGG AGC AAG GCC AAG AGG AGC AGG
F V Y V W K T W G Q Y W Q V
TTC GTG TAC GTG TGG AAG ACC TGG GGC CAG TAC TGG CAG GTG
(SEQ ID NO:68)
靶定的免疫原可以与佐剂和/或细胞因子组合施用以加强免疫应答。示例性佐剂在下面的表III中显示:
表III免疫原性佐剂的类型
佐剂类型 | 一般实例 | 特定实例/参考文献 |
凝胶型 | 氢氧化铝/磷酸铝(“明矾佐剂”) | (Aggerbeck和Heron,1995) |
磷酸钙 | (Relyveld,1986) | |
微生物的 | 胞壁酰二肽(MDP) | (Chedid等人,1986) |
细菌外毒素 | 霍乱毒素(CT)、大肠杆菌不稳定毒素(LT)(Freytag和Clements,1999) | |
基于内毒素的佐剂 | 单磷酰基脂质A(MPL)(Ulrich和Myers,1995) | |
其他细菌的 | CpG寡核苷酸(Corral和Petray,2000)、BCG序列(Krieg,等人Nature,374:576),破伤风类毒素(Rice,等人J.Immunol.,2001,167:1558-1565) | |
微粒 | 可生物降解的聚合物小球体 | (Gupta等人,1998) |
免疫刺激复合物(ISCOM) | (Morein和Bengtsson,1999) | |
脂质体 | (Wassef等人,1994) | |
基于油-乳剂和表面活性剂的佐剂 | 弗氏不完全佐剂 | (Jensen等人,1998) |
微流化(microfluidized)的乳剂 | MF59(Ott等人,1995) | |
SAF(Allison和Byars,1992)(Allison,1999) | ||
皂苷类 | QS-21(Kensil,1996) | |
合成的 | 胞壁酰肽衍生物 | 莫拉丁酯(Lederer,1986)Threony-MDP(Allison,1997) |
非离子嵌段共聚物 | L121(Allison,1999) | |
聚磷腈(PCPP) | (Payne等人,995) | |
合成的多核苷酸 | Poly A:U,Poly I:C(Johnson,1994) | |
沙立度胺衍生物 | CC-4047/ACTIMID (J.Immunol.,168(10):4914-9) |
一种或多种细胞因子也可以是实践本发明中适宜的共刺激组分,作为多肽或者由本发明的组合物内包含的核酸编码(Parmiani,等人Immunol Lett 2000 Sep15;74(1):41-4;Berzofsky,等人NatureImmunol.1:209-219)。适宜的细胞因子包括例如,白细胞介素-2(IL-2)(Rosenberg,等人Nature Med.4:321-327(1998))、IL-4、IL-7、IL-12(Pardoll,1992;Harries,等人J.Gene Med.2000 Jul-Aug;2(4):243-9;Rao,等人J.Immunol.156:3357-3365(1996)综述)、IL-15(Xin,等人Vaccine,17:858-866,1999)、IL-16(Cruikshahk,等人J.Leuk Biol.67(6):757-66,2000)、IL-18(J.Caneer Res.Clin.Oncol.2001.127(12):718-726)、GM-CSF(CSF(Disis,等人Blood,88:202-210(1996))、肿瘤坏死因子-α(TNF-α)或者干扰素-γ(INF-γ)。如本领域已知的,其他细胞因子也可以适于实践本发明。
趋化因子也可以用于辅助诱导或者增强免疫应答。例如,已经表明包含与肿瘤自身抗原融合的CXCL10(IP-10)和CCL7(MCP-3)的融合蛋白诱导抗肿瘤免疫性(Biragyn,等人Nature Biotech.1999,17:253-258)。趋化因子CCL3(MIP-1α)和CCL5(RANTES)(Boyer,等人Vaccine,1999,17(Supp.2):S53-S64)也可以用于实践本发明。其他适宜的趋化因子是本领域已知的。
在一些实施方案中,靶定的免疫原可以用作核酸分子,其单独或者作为递送载体如病毒载体的部分使用。在这些情况中,有利的是组合靶定的免疫原与本发明组合物中的一种或多种共刺激组分,如细胞表面蛋白、细胞因子或者趋化因子。例如共刺激组分可以包括在组合物中作为多肽或者编码该多肽的核酸。适宜的共刺激分子包括,例如,结合CD28家族成员(即,CD28,ICOS;Hutloff,等人Nature1999,397:263-265;Peach,等人J Exp Med 1994,180:2049-2058)的多肽,如CD28结合多肽B7.1(CD80;Schwartz,1992;Chen等人,1992;Ellis,等人J.Immunol.,156(8):2700-9)和B7.2(CD86;Ellis,等人J.Immunol.,156(8):2700-9);结合整联蛋白家族成员(即,LFA-1(CD11a/CD18);Sedwick,等人J Immunol 1999,162:1367-1375;Wülfing,等人Science 1998,282:2266-2269;Lub,等人Immunol Today 1995,16:479-483)、包括ICAM家族的成员(即,ICAM-1、-2或-3)的多肽;结合CD2家族成员(即,CD2,信号传导淋巴细胞激活分子(CDw150或“SLAM”;Aversa,等人J Immunol 1997,158:4036-4044)的多肽,如CD58(LFA-3;CD2配体;Davis,等人Immunol Today 1996,17:177-187)或SLAM配体(Sayos,等人Nature 1998,395:462-469);结合热稳定抗原(HSA或CD24;Zhou,等人Eur J Immunol 1997,27:2524-2528)的多肽;结合TNF受体(TNFR)家族成员(即,4-1BB(CD137;Vinay,等人Semin Immunol 1998,10:481-489))的多肽、OX40(CD134;Weinberg,等人Semin Immunol 1998,10:471-480;Higgins,等人JImmunol 1999,162:486-493)、和CD27(Lens,等人Semin Immunol.1998,10:491-499))如4-1BBL(4-1BB配体;Vinay,等人SeminImmunol 1998,10:481-48;DeBenedette,等人J Immunol 1997,158:551-559)、TNFR结合的因子-1(TRAF-1;4-1BB配体;Saoulli,等人J Exp Med 1998,187:1849-1862,Arch,等人Mol Cell Biol 1998,18:558-565)、TRAF-2(4-1BB和OX40配体;Saoulli,等人J Exp Med1998,187:1849-1862;Oshima,等人Int Immunol 1998,10:517-526,Kawamata,等人J Biol Chem 1998,273:5808-5814)、TRAF-3(4-1BB和OX40配体;Arch,等人Mol Cell Biol 1998,18:558-565;Jang,等人Biochem Biophys Res Commun 1998,242:613-620;KawamataS,等人J Biol Chem 1998,273:5808-5814)、OX40L(OX40配体;Gramaglia,等人J Immunol 1998,161:6510-6517)、TRAF-5(OX40配体;Arch,等人Mol Cell Biol 1998,18:558-565;Kawamata,等人J Biol Chem 1998,273:5808-5814)和CD70(CD27配体;Couderc,等人Cancer Gene Ther.,5(3):163-75)。CD154(CD40配体或“CD40L”;Gurunathan,等人J.Immunol.,1998,161:4563-4571;Sine,等人Hum.Gene Ther.,2001,12:1091-1102)也是适宜的。不同于共刺激分子本身的刺激基序也可以整合入编码TA的核酸中,如CpG基序(Gurunathan,等人Ann.Rev.Immunol.2000,18:927-974)。使用本文描述的试剂和方法,其他刺激基序或共刺激分子也可以用于治疗和/或预防癌症。
这些共刺激组分的任一种可以单独或者与其他试剂组合使用。例如,已经表明CD80、ICAM-1和LFA-3的组合(“TRICOM”)可以加强抗癌免疫应答(Hodge,等人Cancer Res.59:5800-5807(1999)。其他有效的组合包括例如,IL-12+GM-CSF(Ahlers,等人J.Immunol.,158:3947-3958(1997);Iwasaki,等人J.Immunol.158:4591-4601(1997))、IL-12+GM-CSF+TNF-α(Ahlers,等人Int.Immunol.13:897-908(2001))、CD80+IL-12(Fruend,等人Int.J.Cancer,85:508-517(2000);Rao,等人,如前)和CD86+GM-CSF+IL-12(Iwasaki,如前)。本领域技术人员将知道用于实施本发明的额外的组合。
本领域还已知可以阻断抑制性或者负调节免疫机制,从而导致增强的免疫应答。例如,已经表明用抗-CTLA-4(Shrikant,等人Immunity,1996,14:145-155;Sutmuller,等人J.Exp.Med.,2001,194:823-832)、抗-CD25(Sutmuller,如前)、抗-CD4(Matsui,等人J.Immunol.,1999,163:184-193)、融合蛋白IL13Ra2-Fc(Terabe,等人Nature Immunol.,2000,1:515-520)和它们的组合(即,抗-CTLA-4和抗-CD25,Sutmuller,如前)治疗上调抗肿瘤免疫应答。此外,技术人员将知道可以用以调节此类机制的额外试剂或者方法。这些试剂和方法,以及本领域技术人员已知的其他试剂和方法可以用于实践本发明。
表达载体也可以适于用于实践本发明。表达载体通常包含与编码多肽的异源核酸序列(“编码序列”)可操作地连接的侧翼序列。在优选实施方案中,所述多肽由代表靶向序列的第一个氨基酸序列和代表免疫原(即,T细胞表位)的第二个氨基酸序列组成。侧翼序列优选能够实现编码序列的复制、转录和/或翻译并且可操作地连接编码序列。“可操作地连接”指出该核酸序列如此配置从而实现它们的通常功能。例如,当启动子能够指导编码序列转录时,该启动子可操作地连接该编码序列。侧翼序列不必与编码序列邻接,只要它正确发挥功能即可。从而,例如,在启动子序列和编码序列之间可以存在间插的非翻译但是转录的序列,并且仍然可以认为启动子序列可操作地连接编码序列。侧翼序列可以是同源的(即,来自与宿主细胞相同的物种和/或株)、异源的(即,来自不同于宿主细胞物种或者株的物种)、杂合的(即,来自一种以上来源的侧翼序列的组合),或者合成的。侧翼序列还可以是正常发挥功能以调节宿主基因组中编码多肽的核苷酸序列的表达的序列。
在某些实施方案中,优选侧翼序列是转录调节区,其驱动靶细胞中高水平的基因表达。转录调节区可以包含例如,启动子、增强子、沉默子、阻遏元件,或者它们的组合。转录调节区可以是组成型的或者组织-或者细胞类型特异性的(即,该区域在一种类型的组织或者细胞中与另一种类型的组织或者细胞中相比驱动更高水平的转录)。同样地,转录调节区的来源可以是任意原核或者真核生物、任何脊椎动物或者无脊椎动物,或者任何植物,前提是侧翼序列在宿主细胞机器中是有功能的并且可以由宿主细胞机器激活。在本发明的实践中可以利用多种转录调节区。
适宜的转录调节区除其他的以外包括CMW启动子(即,CMV立即早期启动子);来自真核生物基因(即,雌激素诱导型鸡卵清蛋白基因、干扰素基因、糖皮质激素诱导型酪氨酸氨基转换酶基因和胸苷激酶基因)的启动子;和主要早期和晚期腺病毒基因启动子;SV40早期启动子区(Bernoist和Chambon,1981,Nature 290:304-10);劳斯肉瘤病毒(RSV)的3’长末端重复序列(LTR)中所含的启动子(Yamamoto,等人,1980,Cell 22:787-97);单纯疱疹病毒胸苷激酶(HSV-TK)启动子(Wagner等人,1981,Proc.Natl.Acad.Sci.U.S.A.78:1444-45);金属硫蛋白基因的调节序列(Brinster等人,1982,Nature 296:39-42);原核生物表达载体,如β-内酰胺酶启动子(Villa-Kamaroff等人,1978,Proc.Natl.Acad.Sci.U.S.A.,75:3727-31);或tac启动子(DeBoer等人,1983,Proc.Natl.Acad.Sci.U.S.A.,80:21-25)。组织和/或细胞类型特异性转录控制区包括,例如,在胰腺腺泡细胞中有活性的弹性蛋白酶I基因控制区(Swift等人,1984,Cell 38:639-46;Ornitz等人,1986,Cold Spring HarborSymp.Quant.Biol.50:399-409(1986);MacDonald,1987,Hepatology7:425-515);在胰腺β细胞中有活性的胰岛素基因控制区(Hanahan,1985,Nature 315:115-22);在淋巴样细胞中有活性的免疫球蛋白基因控制区(Grosschedl等人,1984,Cell 38:647-58;Adames等人,1985,Nature 318:533-38;Alexander等人,1987,Mol.Cell.Biol.,7:1436-44);睾丸细胞、乳腺细胞、淋巴样细胞和肥大细胞中的小鼠乳癌病毒控制区(Leder等人,1986,Cell 45:485-95);肝脏中的清蛋白基因控制区(Pinkert等人,1987,Genes and Devel.1:268-76);肝脏中的甲胎蛋白基因控制区(Krumlauf等人,1985,Mol.Cell.Biol.,5:1639-48;Hammer等人,1987,Science 235:53-58);肝脏中的α-1抗胰蛋白酶基因控制区(Kelsey等人,1987,Genes and Devel.1:161-71);髓样细胞中的β-珠蛋白基因控制区(Mogram等人,1985,Nature 315:338-40;Kollias等人,1986,Cell 46:89-94);脑中的少突胶质细胞中的髓鞘碱性蛋白基因控制区(Readhead等人,1987,Cell 48:703-12);骨骼肌中肌球蛋白轻链-2基因控制区(Sani,1985,Nature 314:283-86);和下丘脑中促性腺激素释放激素基因控制区(Mason等人,1986,Science 234:1372-78),和黑素瘤细胞中的酪氨酸酶启动子(Hart,I.Semin Oncol 1996 Feb;23(1):154-8;Siders,等人Cancer Gene Ther 1998 Sep-Oct;5(5):281-91)。本领域已知其他适宜的启动子。
编码靶定的免疫原的核酸分子可以作为病毒和非病毒载体的部分施用。在一个实施方案中,用DNA载体向患者递送编码靶定的免疫原和/或相关分子(即,共刺激分子、细胞因子或者趋化因子)的核酸。在这种情况下,可以用多种策略提高此类机制的效率,包括,例如,使用自我复制性病毒复制子(Caley,等人1999.Vaccine,17:3124-2135;Dubensky,等人2000.Mol.Med.6:723-732;Leitner,等人2000.Cancer Res.60:51-55)、密码子最优化(Liu,等人2000.Mol.Ther.,1:497-500;Dubensky,如前;Huang,等人2001.J.Virol.75:4947-4951)、体内电穿孔(Widera,等人2000.J.Immunol.164:4635-3640)、整合编码共刺激分子、细胞因子和/或趋化因子的核酸(Xiang,等人1995.Immunity,2:129-135;Kim,等人1998.Eur.J.Immunol.,28:1089-1103;Iwasaki,等人1997.J.Immunol.158:4591-4601;Sheerlinck,等人2001.Vaccine,19:2647-2656)、整合刺激基序,如CpG(Gurunathan,如前;Leiter,如前)、靶向内吞的或者遍在蛋白加工途径的序列(Thomson,等人1998.J.Virol.72:2246-2252;Velders,等人2001.J.Immunol.166:5366-5373)、引发-加强方案(prime-boost regimens)(Gurunathan,如前;Sullivan,等人2000.Nature,408:605-609;Hanke,等人1998.Vaccine,16:439-445;Amara,等人2001.Science,292:69-74)、蛋白酶体敏感切割位点,和使用粘膜递送载体,如沙门氏菌(Salmonella)(Darji,等人1997.Cell,91:765-775;Woo,等人2001.Vaccine,19:2945-2954)。其他方法是本领域已知的,一些方法在下文描述。
已经成功用于将核酸导入宿主的多种病毒载体除其他的以外包括逆转录病毒、腺病毒、腺伴随病毒(AAV)、疱疹病毒和痘病毒.本领域将理解本领域可以利用许多此类病毒载体.用本领域技术人员普遍可得到的标准重组技术可以构建本发明的载体。此类技术可以见普通分子生物学参考文献,如Molecular Cloning:A LaboratoryManual(Sambrook,等人,1989,Cold Spring Harbor LaboratoryPress)、Gene Expression Technology(Methods in Enzymology,Vol.185,编者:D.Goeddel,1991.Academic Press,San Diego,CA),和PCR Protocols:A Guide to Methods and Applications(Innis,等人1990.Academic Press,San Diego,CA)。
优选的逆转录病毒载体是慢病毒衍生物以及鼠或者鸟类逆转录病毒的衍生物。适宜的逆转录病毒载体的实例包括例如,莫洛尼鼠白血病病毒(MoMuLV)、Harvey鼠肉瘤病毒(HaMuSV)、鼠乳癌病毒(MuMTV)、SIV、BIV、HIV和劳斯肉瘤病毒(RSV)。许多逆转录病毒载体可以整合多种外源核酸序列。由于重组逆转录病毒是有缺陷的,所以它们需要帮助来产生感染性病毒颗粒。该帮助可以由例如编码逆转录病毒结构基因的辅助细胞系提供。适宜的辅助细胞系除其他的以外包括ψ2、PA317和PA12。用此类细胞系产生的载体病毒体可以用于感染组织细胞系,如NIH 3T3细胞,以产生大量嵌合的逆转录病毒病毒体。可以通过常规方法(即,注射)或者通过将“生产者细胞系”植入靶细胞群体附近来施用逆转录病毒载体(Culver,K.,等人,1994,Hum.Gene Ther.,5(3):343-79;Culver,K.,等人,Cold Spring Harb.Symp.Quant.Biol.,59:685-90);Oldfield,E.,1993,Hum.Gene Ther.,4(1):39-69)。将生产者细胞系工程改造以产生病毒载体和在靶细胞的附近释放病毒颗粒。一部分释放的病毒颗粒与靶细胞接触并感染那些细胞,从而向靶细胞递送本发明的核酸。注射靶细胞后,发生载体核酸的表达。
腺病毒载体已经被证明尤其可用于向真核细胞的基因转移(Rosenfeld,M.,等人,1991,Science,252(5004):431-4;Crystal,R.,等人,1994,Nat.Genet.,8(1.):42-51)、研究真核基因表达(Levrero,M.,等人,1991,Gene,101(2):195-202)、疫苗开发(Graham,F.和Prevec,L.,1992,Biotechnology,20:363-90)和用于动物模型中(Stratford-Perricaudet,L.,等人,1992,BoneMarrow Transplant.,9(Suppl.1):151-2;Rich,D.,等人,1993,Hum.Gene Ther.,4(4):461-76)。对不同组织体内施用重组Ad的实验途径除其他的以外包括气管内滴注(Rosenfeld,M.,等人,1992,Cell,68(1):143-55)、注射到肌肉(Quantin,B.,等人,1992,Proc.Natl.Acad.Sci.U.S.A.,89(7):2581-4)、外周静脉内注射(Herz,J.,和Gerard,R.,1993,Proc.Natl.Acad.Sci.U.S.A.,90(7):2812-6)和趋实体(stereotactic)接种到脑(Le Gal La Salle,G.,等人,1993,Science,259(5097):988-90)。
腺伴随病毒(AAV)表现出高水平感染性、宽宿主范围和整合到宿主细胞基因组中的特异性(Hermonat,P.,等人,1984,Proc.Natl.Acad.Sci.U.S.A.,81(20):6466-70)。1型单纯疱疹病毒(HSV-1)是另一种吸引人的载体系统,由于它的神经营养性质而特别用于神经系统中(Geller,A.,等人,1991,Trends Neurosci.,14(10):428-32;Glorioso,等人,1995,Mol.Biotechnol.,4(1):87-99;Glorioso,等人,1995,Annu.Rev.Microbiol.,49:675-710)。
痘病毒是另一种有用的表达载体(Smith,等人1983,Gene,25(1):21-8;Moss,等人,1992,Biotechnology,20:345-62;Moss,等人,1992,Curr.Top.Microbiol.Immunol.,158:25-38;Moss,等人1991.Science,252:1662-1667)。已表明有用的痘病毒除其他的以外包括牛痘、NYVAC、禽痘(avipox)、禽痘、金丝雀痘、ALVAC和ALVAC(2)。
NYVAC(vP866)来自痘苗病毒的哥本哈根(Copenhagen)疫苗株,这通过缺失基因组的编码已知的或者潜在毒力因子的6个非必需区得到(见例如,美国专利号5,364,773和5,494,807).还将缺失基因座工程改造为用于插入外来基因的受体基因座。缺失的区域为:胸苷激酶基因(TK;J2R)vP410;出血区(u;B13R+B14R)vP553;A型内含体区(ATI;A26L)vP618;血凝素基因(HA;A56R)vP723;宿主范围基因区(C7L-K1L)vP804;和大亚基核糖核苷酸还原酶(14L)vP866。NYVAC是基因工程化的痘苗病毒株,其通过特异缺失编码与毒性和宿主范围相关的基因产物的18个可读框产生。已经表明NYVAC可用于表达TA(见,例如,美国专利号6,265,189)。根据布达佩斯条约(Budapest Treaty),NYVAC(vP866)、vP994、vCP205、vCP1433、placZH6H4Lreverse、pMPC6H6K3E3和pC3H6FHVB保藏在ATCC,保藏号分别为VR-2559、VR-2558、VR-2557、VR-2556、ATCC-97913、ATCC-97912和ATCC-97914。
基于ALVAC的重组病毒(即,ALVAC-1和ALVAC-2)也适合用于实践本发明(见,例如,美国专利号5,756,103)。除了ALVAC(2)基因组包含处于牛痘启动子控制下的牛痘E3L和K3L基因外,ALVAC(2)与ALVAC(1)相同(美国专利号6,130,066;Beattie等人,1995a,1995b,1991;Chang等人,1992;Davies等人,1993)。已经证明ALVAC(1)和ALVAC(2)都可以用于表达外源DNA序列,如TA(Tartaglia等人,1993a,b;美国专利号5,833,975)。ALVAC已经根据布达佩斯条约保藏在美国典型培养物保藏中心(ATCC),10801 University Boulevard,Manassas,Va.20110-2209,美国,ATCC保藏号为VR-2547。
另一种有用的痘病毒载体是TROVAC。TROVAC是指减毒的禽痘,其是从禽痘病毒的FP-1疫苗株得到的噬菌斑克隆的分离物,所述疫苗株已经许可用于接种1天龄的小鸡。TROVAC同样根据布达佩斯条约保藏在ATCC,保藏号2553。
“非病毒”质粒载体也可以适于某些实施方案。优选的质粒载体与细菌、昆虫和/或哺乳动物宿主细胞相容。此类载体包括,例如,PCR-II、pCR3和pcDNA3.1(Invitrogen,San Diego,CA)、pBSII(Stratagene,La Jolla,CA)、pET15(Novagen,Madison,WI)、pGEX(Pharmacia Biotech,Piscataway,NJ)、pEGFP-N2(Clontech,Palo Alto,CA)、pETL(BlueBacII,Invitrogen)、pDSR-α(PCT公开号WO 90/14363)和pFastBacDual(Gibco-BRL,Grand Island,NY)以及Bluescript质粒衍生物(高拷贝数的基于COLE1-的噬菌粒,Stratagene Cloning Systems,La Jolla,CA)、设计用于克隆Taq-扩增的PCR产物的PCR克隆质粒(例如,TOPOTM TA cloning试剂盒、PCR2.1质粒衍生物,Invitrogen,Carlsbad,CA)。细菌载体也可以用于本发明。这些载体包括例如,志贺氏菌属(Shigella)、沙门氏菌属(Salmonella)、霍乱弧菌(Vibrio cholerae)、乳杆菌属(Lactobacillus)、卡介苗(BCG)和链球菌属(Streptococcus)(见例如,WO 88/6626;WO 90/0594;WO 91/13157;WO 92/1796;和WO 92/21376)。许多其他非病毒质粒表达载体和系统是本领域已知的并且可以用于本发明。
其他递送技术也可以足够实施本发明,所述技术包括例如,DNA-配体复合体、腺病毒-配体-DNA复合体、直接注射DNA、CaPO4沉淀、基因枪技术、电穿孔和胶态分散体系统。胶态分散体系统包括大分子复合体、纳米胶囊、小球体、珠和基于脂质的系统,包括水包油乳剂、微团、混合微团和脂质体。本发明的优选的胶态系统是脂质体,其是用作体外和体内递送载体的人工膜囊。RNA、DNA和完整病毒体可以被囊化在水性内部并以生物活性形式递送到细胞(Fraley,R.,等人,1981,Trends Biochem.Sci.,6:77)。脂质体的组成通常是磷脂,特别是高相变温度磷脂的组合,通常与类固醇,特别是胆固醇组合。也可以使用其他磷脂或者脂质。脂质体的物理特征取决于pH、离子强度和二价阳离子的存在。用于脂质体生产的脂质的实例包括磷脂酰基化合物,如磷脂酰甘油、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰乙醇胺、鞘脂、脑苷脂和神经节苷脂。尤其有用的是二酰基磷脂酰甘油,其中脂质部分含有14-18个碳原子,尤其16-18个碳原子,并且是饱和的。阐明性磷脂包括卵磷脂酰胆碱、二棕榈酰磷脂酰胆碱和二硬脂酰磷脂酰胆碱。
可以用本领域技术人员已知的多种技术的任一种完成本发明的靶定的免疫原向宿主的施用。可以根据药剂学的常规方法加工包含靶定的免疫原的组合物以产生药物试剂用于施用于患者,包括人和其他哺乳动物(即,产生“药物组合物”)。优选以含有例如给定量的DNA、病毒载体颗粒、多肽或者肽的剂量单位的形式制备药物组合物。用于人或者其他哺乳动物的适宜的日剂量可以依赖于患者的状况和其他因素而变,但是再次可以用常规方法确定。
药物组合物可以经口、肠胃外、通过吸入喷雾、直肠或者局部地以含有常规可药用载体、佐剂和媒介物的剂量单位制剂施用。本文所用的术语“可药用载体”或者“生理学可接受的载体”是指适于作为药物组合物完成或者增强核酸、多肽或者肽的递送的一种或多种制剂材料。“药物组合物”是包含治疗有效量的核酸或多肽的组合物。术语“有效量”和“治疗有效量”各自指用于诱导或者增强有效免疫应答的核酸或者多肽的量。优选本发明的组合物提供宿主中抗肿瘤免疫应答的诱导或者增强,它保护宿主免于患肿瘤和/或允许宿主从身体消除现有肿瘤。
对于经口施用,药物组合物可以是几种形式的任一种,其中包括,例如,胶囊、片剂、悬浮液或者液体。可以作为与适宜的载体(包括盐水、葡萄糖或者水)的组合物注射施用液体。本文所用的术语肠胃外的包括皮下、静脉内、肌内、鼻内、输注或者腹膜内施用。用于经直肠施用药物的栓剂可以通过将药物与适宜的在常温下为固体但是在直肠温度下为液体的非刺激性赋形剂如可可脂和聚乙二醇混合来制备。
用本发明的组合物免疫宿主或者另外治疗病症或者疾病的剂量方案基于多种因素,包括患者的疾病类型、年龄、体重、性别、医学状况、状况的严重性、施用途径和所用的具体化合物。从而,剂量方案可以有很大变动,但是可以用标准方法常规地确定。
尽管本发明的组合物可以作为唯一活性药剂施用,但是它们也可以与一种或多种其他组合物或者试剂组合使用。当作为组合施用时,可以将单独组分配制为在相同或不同时间施用的分开的组合物,或者可以将各组分组合为一种组合物。
还提供了包含本发明的组合物的试剂盒。试剂盒可以包含单独的容器,其含有适宜的载体、稀释剂或者赋形剂。试剂盒还可以包括额外的抗癌、抗肿瘤或者抗瘤剂和/或减小或者减轻抗瘤、抗肿瘤或者抗癌剂的不良作用的试剂用于共同或者顺序施用。此外,试剂盒可以包括关于混合或组合各成分和/或关于施用的说明书。
从为了阐明而给出的下面的实施例将更好的理解本发明和它的许多优点。
实施例
实施例1
制备免疫原性靶肽
通过Bio-Synthesis Incorporated(Lewisville,Texas)用标准技术合成所有肽。
为了证明表位缀合系统的可行性,将细胞毒性T淋巴细胞(CTL)表位缀合到多种转导序列。选择下面的转胞吞肽用于连接到表位:
TAT: GYGRKKRRQRRR
hPER1-1:SRRHHCRSKAKRSRHH
hPER1-2:RRHHRRSKAKRSR
AntPHD: RQIKIWFQNRRMKWKK
某些表位肽用接头序列连接到转胞吞序列。从天然发现直接处于表位序列的N-末端的序列选择接头,或者基于已知的免疫学参数选择接头。所选的接头序列在下面显示:
OVA: LEQLE(天然的)
DEVWEL(合成的)
NP 366-374: RGVQI(天然的)
gp100(154-162): FVYVW(天然的)
选择一些表位,如下所示:
OVA: SIINFEKL
NP 366-374: ASNENMETM
(Rotzschke et al.1990
Nature 348:252)
gp100(280-288(9V)): YLEPGPVTV
(Parkhurst et al.1996
J.Immunol.157:2539)
gp100(154-162): KTWGQYWQV
(Kawakami et al.1995.
J.Immunol.154:3961)
然后通过组合上述转胞吞肽、接头序列和表位肽合成几种免疫原性靶,如下所示:
TAT-OVA肽:
GYGRKKRRQRRR-SIINFEKL
GYGRKKRRQRRR-LEQLE-SIINFEKL
GYGRKKRRQRRR-DEVWEL-SIINFEKL
hPER1-OVA肽:
RRHHRRSKAKRSRSIINFEKL
RRHHRRSKAKRSR-LEQLE-SIINFEKL
RRHHRRSKAKRSR-SGQL-SIINFEKL
RRHHRRSKAKRSR-DEVWEL-SIINFEKL
RRHHRRSKAKRSR-FVYVW-SIINFEKL
hPER1-NP肽
RRHHRRSKAKRSR-ASNENMETM
RRHHRRSKAKRSR-RGVQI-ASNENMETM
RRHHRRSKAKRSR-FVYVW-ASNENMETM
hPER1-1-gp100(280-288)
SRRHHCRSKAKRSRHH-YLEPGPVTV
hPER1-2-gp100(154-162)
RRHHRRSKAKRSR-KTWGQYWQV
RRHHRRSKAKRSR-FVYVW-KTWGQYWQV
AntPHD-gp100
RQIKIWFQNRRMKWKK-KTWGQYWQV
RQIKIWFQNRRMKWKK-FVYVW-KTWGQYWQV
然后在免疫学测定中测试这些肽,如下文所述。
实施例2
免疫学测试
A.hPER1-CTL表位缀合物当与细胞体外温育时可以形成CTL靶结构。
为了确定hPER1-CTL缀合物是否可以形成CTL靶结构,将51Cr标记的RMA细胞用10-11g/ml NP肽(ASNENMETM)或hPER1-NP肽(RRHHRRSKAKRSRASNENMETM)脉冲,或者不处理(无肽)并在37℃温育1小时。然后洗涤细胞并在标准的4小时铬释放测定中测试CTL识别,其中使用从用流感病毒免疫的C57BL/6小鼠的脾脏得到的T细胞。图1A证明RMA靶细胞当与10pg/ml hPER1-NP肽温育时可以对于CTL-介导的裂解敏化。
此外,将51Cr标记的P815-A2/Kb细胞用10-6g/ml 280-9V肽(YLEPGPVTV)或hPER1-280-9V(RRHHRRSKKAKRSRYLEPGPVTV)脉冲,或者不处理(无肽)并在37℃温育1小时。然后洗涤细胞并在标准的4小时铬释放测定中测试CTL识别,其中使用从用不完全弗氏佐剂中的280-9V肽免疫的HLA-A2/Kb转基因小鼠的脾脏得到的T细胞。如所指出的,在该测定中包括5μg/ml布雷菲德菌素A(BFA)以阻断新生的I类MHC分子的表面表达。图1B证明用10-6g/mlhPER1-280-9V肽可以敏化P815-A2/Kb靶细胞。如果用布雷菲德菌素A处理hPER1-280-9V脉冲的靶细胞,则CTL杀伤水平降低,所述布雷菲德菌素A阻断新合成的MHC分子的细胞内转运。
这些实验证明hPER1介导的细胞内递送提供了鼠T细胞的增加的敏化。同样地,进行实验来证明在人CTL中的该效果。
B.hPER1-CTL表位缀合物在人T细胞培养系统中是免疫原性的
在IL-2(50U/ml)、IL-7(10ng/ml)、LPS(10μg/ml)、CD40-配体表达性3T3细胞和肽(10μg/ml 280-9V或hPER1-280-9V)的存在下培养来自HLA-A2-阳性患者的外周血单核细胞(PBMC)。在第11、22和32天,通过在IL-2(50U/ml)和IL-7(10ng/ml)的存在下进行培养来再次刺激细胞并用肽(100μg/ml 280-9V或hPER1-280-9V)对自体的CD40-配体激活的PBMC脉冲3小时。在第42天,在标准铬释放测定中测试培养物的CTL活性,其中使用用280-9V肽或者对照A2-结合肽脉冲的C1R-A2靶细胞。图2证明通过用hPER1-280-9V重复体外刺激可以诱导280-9V特异的人CTL。
C.不存在佐剂时hPER1-CTL表位肽缀合物在体内是免疫原性的
图3证明了在I-Ab-限制的T辅助表位(100μg)存在下,用100μg154、hPER1-154、280-9V或hPER1-280-9V皮下免疫HLA-A2/Kb转基因小鼠(每组4只)的结果。在第14和28天类似地加强免疫小鼠。在第42天,用适当的野生型肽体外单独再刺激脾细胞(每组2只小鼠)6天,然后通过ELISPOT测试IFN-γ分泌(图3A)或用肽脉冲的C1R-A2细胞进行CTL测定(图3B)。在第57天,类似地测试每组中剩余小鼠。显示了来自每组的平均应答。
图3A证明不存在佐剂时,用hPER1-154(加上T-辅助肽)免疫HLA-A2/Kb转基因小鼠可以诱导154-特异的IFN-γ应答。用野生型亲本肽进行类似免疫不能诱导应答。如图3B中所示,通过用hPER1-154或者hPER1-280-9V免疫可以诱导肽特异的CTL应答,而用野生型亲本肽免疫后没有诱导应答。
成熟的树突细胞(DC)是有效的抗原呈递细胞,已经表明它们在小鼠中静脉内注射后产生有力的CTL应答。因此,我们测试了转胞吞肽在基于DC的疫苗的背景下产生CTL应答的能力。来自鼠骨髓的树突细胞在体外成熟,仅用SIINFEKL或者用或者不用接头缀合到Tat或者hPER1的SIINFEKL脉冲,并在C57BL/6小鼠的尾静脉中静脉内注射。免疫后一周,体外再次刺激后,测试来自接种动物的脾细胞的CTL活性。如图4中所示,所有SIINFEKL脉冲的DC都能够产生有力的CTL应答,而用不相关的肽(TRP2)脉冲的DC是非免疫原性的。用hPER1-OVA脉冲的DC比用天然SIINFEKL肽或者hPER1-LEQLE-SIINFEKL脉冲的DC产生更强的应答。类似地,TAT-LEQLE-SIINFEKL肽比没有接头的TAT-SIINFEKL的免疫原性更低,这与下述体外观察相一致。
此外,在HLA-A2/Kb转基因小鼠(Sherman品系)仅用gp100-154肽或者用或不用接头FVYVW缀合到hPER1或AntpHD的gp100-154皮下免疫后,评估所述小鼠中的CTL应答。在第21和42天加强免疫小鼠,并在第63天从接种的动物收获脾细胞并在体外再次刺激5天后测试CTL活性。如图5中所示,仅154肽甚至在不完全弗氏佐剂的存在下也不能产生有效CTL应答。当与AntpHD-154或hPER1-154结合时,观察到弱应答,其随着接头序列FVYVW的存在而增加。然而,当表位缀合到hPER1和接头序列FVYVW时,观察到最有力的活性。
在实施图6和13中描述的实验中,通过特定途径用50纳摩尔(如果没有另外指出)肽加上50纳摩尔小鼠中的乙型肝炎表位作为辅助CD4肽,对小鼠进行免疫。第一次注射后3周,用相同的方案进行加强,且之后三周,收获脾脏并将其匀浆为单一悬浮液。将全部脾细胞与0.5μg/ml表位肽置于培养物中并在37℃温育5天。Ficoll处理以纯化活细胞后在培养的第5天进行CTL测定。所用的对照是匹配的Kb或者A2结合肽。结果证明这些靶定的免疫原当皮内、皮下或者鼻内施用时诱导免疫应答(图6)。
图7中的结果证明i)Tat和hPer1都可以比仅用该肽诱导更高水平的CTL,和ii)与Tat转导序列相比,hPER1转导序列至少优于OVA肽SIINFEKL。如图7中所示,在所有测试的E∶T比率下施用hPER1-DEVWEL-SIINFEKL与Tat-DEVWEL-SIINFEKL相比诱导更高水平的细胞毒性。
如图8中所示,辅助CD4乙型肝炎肽的包括在一些情况中对于用免疫原性靶产生免疫性是重要的。在辅助肽的存在下用hPER1-FVYVW-154肽接种小鼠诱导显著的T细胞细胞毒性。不存在辅助肽时接种诱导低得多水平的细胞毒性。有趣的是,如图9中所示,增加免疫原性靶的量克服了对辅助肽的依赖性。
图10证明不存在佐剂时施用的靶定的免疫原与和佐剂一起施用未缀合的肽一样有效。不用佐剂,皮下施用免疫原性靶hPER1-FVYVW-SIINFEKL和hPER1-DEVWEL-SIINFEKL。将OVA肽(SIINFEKL)与不完全弗氏佐剂一起施用。如图所示,免疫原性靶和IFA中的OVA肽的细胞毒性水平是相当的。此外,接头序列的性质可以显著增加产生CTL的潜能或者能力。而接头FVYWV是最佳接头,接头DEVWEL及然后是SGQL诱导较低水平的细胞毒性。这些观察表明接头的性质是体内诱导CTL的重要因素。
D.hPER1表位缀合延长了肽呈递和免疫应答
为进一步研究CTL表位偶联到hPER1转导结构域的效果,开发了下面的体外测定来评估细胞与肽温育后抗原呈递的动力学。在图11中,来自C57BL/6小鼠的脾细胞与不同的基于OVA的肽在37℃温育1小时。然后洗涤细胞以除去任何残留的游离肽,并在培养基中37℃下温育0、4、8、24或者30小时。然后通过ELISPOT测试细胞刺激从SIINFEKL特异的T细胞产生IFN-γ的能力。结果表明用天然OVA肽脉冲的细胞在24小时时失去了它们的刺激能力,而用hPER1-SGQL-SIINFEKL或TAT-DEVWEL-SIINFEKL脉冲的细胞甚至在30小时后活性也没有减小。不存在接头序列时,OVA与hPER1或者TAT的缀合也相对于天然OVA肽增强了抗原呈递,尽管它们的活性低于含有定制设计的接头的肽。掺入作为接头的天然OVA侧翼序列(LEQLE)的hPER1和TAT缀合物没有显示出相对于天然肽的改善。
图12阐明了用NP系统进行的相似分析。这里,天然NP肽在24小时温育后显示出活性损失。然而,用hPER1-NP或hPER1-RGVQI-NP肽脉冲的细胞保持它们刺激T细胞的能力5天以上,这是该测定的极限。总之,这些数据证明hPER1可以延长抗原呈递持续时间,并且可以通过适当接头的设计进一步最优化。
体内实验进一步证实靶定的免疫原能够诱导持久的免疫学记忆。如图13中所示,仅用154肽免疫在施用后3周或者3个月时不诱导细胞毒性。相反,hPER1-FVYVW-154诱导细胞毒性,其可以在施用后至少3个月都是可检测的。该结果表明施用靶定的免疫原与免疫记忆应答有关,而未缀合的肽却并非这样。
表IV概述了在小鼠中进行的免疫原性实验。从此处给出的结果可以得出免疫原性靶可以用于产生特异并且强烈的免疫应答。
表IV体内免疫原性研究概述
转胞吞序列 | 接头 | 肽 | 序列 | 小鼠中的免疫原性 |
- | - | gp100-154 | KTWGQYWQV | - |
AntpHD | FVYVW | gp100-154 | RQIKIWFQNRRMKWKKFVYVWKTWGQYWQV | ++++ |
AntpHD | L | gp100-154 | RQIKIWFQNRRMKWKKLKTWGQYWQV | +++ |
AntpHD | - | gp100-154 | RQIKIWFQNRRMKWKKTWGQYWQV | + |
hPER1 | - | gp100-154 | GRRHHRRSKAKRSRKTWGQYWQV | + |
hPER1 | FVYVW | gp100-154 | RRHHRRSKAKRSRFVYVWKTWGQYWQV | ++++ |
- | - | flu NP366-374 | ASNENMETM | - |
hPER1 | - | flu NP366-374 | GRRHHRRSKAKRSRASNENMETM | - |
hPER1 | RGVQI | flu NP366-374 | RRHHRRSKAKRSRRGVQIASNENMETM | - |
hPER1 | L | flu NP366-374 | RRHHRRSKAKRSRLASNENMETM | - |
hPER1 | FVYVW | flu NP366-374 | RRHHRRSKAKRSRLASNENMETM | ++ |
- | - | TRP2 | SVYDFFVWL | ++ |
hPER1 | - | TRP-2 | RRHHRRSKAKRSRSVYDFFVWL | ++ |
hPER1 | FVYVW | TRP-2 | RRHHRRSKAKRSRFVYVWSVYDFFVWL | +++ |
hPER1 | L | TRP2 | RRHHRRSKAKRSRLSVYDFFVWL | ++ |
- | - | OVA(SIINFEKL) | SIINFEKL | - |
tat | DEVWEL | OVA(SIINFEKL) | YGRKKRRQRRRDEVWELSIINFEKL | +++ |
hPER1 | - | OVA(SIINFEKL) | RRHHRRSKAKRSRSIINFEKL | + |
hPER1 | SGQL | OVA(SIINFEKL) | RRHHRRSKAKRSRSGQLSIINFEKL | + |
hPER1 | DEVWEL | OVA(SIINFEKL) | RRHHRRSKAKRSRDEVWELSIINFEKL | ++++ |
hPER1 | FVYVW | OVA(SIINFEKL) | RRHHRRSKAKRSRFVYVWSIINFEKL | ++++ |
hPER1 | L | OVA(SIINFEKL) | RRHHRRSKAKRSRLSIINFEKL | + |
序列表
TAT: GYGRKKRRQRRR (SEQ ID NO.:1)
AntP: RQIKIWFQNRRMKWKK (SEQ ID NO.:2)
PER1-1: SRRHHCRSKAKRSRHH (SEQ ID NO.:3)
PER1-2: RRHHRRSKAKRSR (SEQ ID NO.:4)
gp100-280-288(9V) YLEPGPVTV (SEQ ID NO:5)
gp100-154-162 KTWGQYWQV (SEQ ID NO:6)
MART-1 32 ILTVILGVL (SEQ.ID.NO.7)
MART-1 31 GILTVILGV (SEQ.ID.NO.8)
MART-1 99 NAPPAYEKL (SEQ.ID.NO.9)
MART-1 1 MPREDAHFI (SEQ.ID.NO.10)
MART-1 56 ALMDKSLHV (SEQ.ID.NO.11)
MART-1 39 VLLLIGCWY (SEQ.ID.NO.12)
MART-1 35 VILGVLLLI (SEQ.ID.NO.13)
MART-1 61 SLHVGTQCA (SEQ.ID.NO.14)
MART-1 57 LMDKSLHVG (SEQ.ID.NO.15)
MAGE-A3 115 ELVHFLLLK (SEQ ID NO:16)
MAGE-A3 285 KVLHHMVKI (SEQ ID NO:17)
MAGE-A3 276 RALVETSYV (SEQ ID NO:18)
MAGE-A3 105 FQAALSRKV (SEQ ID NO:19)
MAGE-A3 296 GPHISYPPL (SEQ ID NO:20)
MAGE-A3 243 KKLLTQHFV (SEQ ID NO.21)
MAGE-A3 24 GLVGAQAPA (SEQ ID NO.22)
MAGE-A3 301 YPPLHEWVL (SEQ ID NO.23)
MAGE-A3 71 LPTTMNYPL (SEQ ID NO.24)
Tyr 171 NIYDLFVWM (SEQ ID NO:25)
Tyr 444 DLGYDYSYL (SEQ ID NO:26)
Tyr 57 NILLSNAPL (SEQ ID NO:27)
TRP-1 245 SLPYWNFAT (SEQ ID NO:28)
TRP-1 298 TLGTLCNST (SEQ ID NO:29)
TRP-1 481 IAVVGALLL (SEQ ID NO:30)
TRP-1 181 NISIYNYFV (SEQ ID NO:31)
TRP-1 439 NMVPFWPPV (SEQ ID NO:32)
TAT(SEQ ID NO.:33):
GGCTACGGCAGGAAGAAGAGGAGGCAGAGGAGGAGG
AntP(SEQ ID NO.:34):
AGGCAGATCAAGATCTGGTTCCAGAACAGGAGGATGAAGTGGAAGAAG
PER1-1(SEQ ID NO.:35):
AGCAGGAGGCACCACTGCAGGAGCAAGGCCAAGAGGAGCAGGCACCAC
PER1-2(SEQ ID NO.:36):
GGCAGGAGGCACCACAGGAGGAGCAAGGCCAAGAGGAGCAGG
gp100-280-288(9V)(SEQ ID NO.:37):
TACCTGGAGCCCGGCCCCGTGACCGTG
gp100-154-162(SEQ ID NO.:38):
AAGACCTGGGGCCAGTACTGGCAGGTG
MART-1 32: ATCCTGACAGTGATCCTGGGAGTCTTA (SEQ ID NO:39)
MART-1 31: GGCATCCTGACAGTGATCCTGGGAGTC (SEQ ID NO:40)
MART-1 99: AATGCTCCACCTGCTTATGAGAAACTC (SEQ ID NO:42)
MART-1 1: ATGCCAAGAGAAGATGCTCACTTCATC (SEQ ID NO:43)
MART-1 56: GCCTTGATGGATAAAAGTCTTCATGTT (SEQ ID NO:44)
MART-1 39: GTCTTACTGCTCATCGGCTGTTGGTAT (SEQ ID NO:45)
MART-1 35: GTGATCCTGGGAGTCTTACTGCTCATC (SEQ ID NO:46)
MART-1 61: AGTCTTCATGTTGGCACTCAATGTGCC (SEQ IDNO:47)
MART-1 57: TTGATGGATAAAAGTCTTCATGTTGGC (SEQ ID NO:48)
MAGE-A3 115:GAGTTGGTTCATTTTCTGCTCCTCAAG (SEQ ID NO.49)
MAGE-A3 285:AAAGTCCTGCACCATATGGTAAAGATC (SEQ.ID.NO.50)
MAGE-A3 276:AGGGCCCTCGTTGAAACCAGCTATGTG (SEQ ID.NO.51)
MAGE-A3 105:TTCCAAGCAGCACTCAGTAGGAAGGTG (SEQ ID.NO.52)
MAGE-A3 296:GGACCTCACATTTCCTACCCACCCCTG (SEQ.ID.NO.53)
MAGE-A3 243:AAGAAGCTGCTCACCCAACATTTCGTG (SEQ ID.NO.54)
MAGE-A3 24: GGCCTGGTGGGTGCGCAGGCTCCTGCT (SEQ ID NO:55)
MAGE-A3 301:TACCCACCCCTGCATGAGTGGGTTTTG (SEQ ID.NO.56)
MAGE-A3 71: CTCCCCACTACCATGAACTACCCTCTC(SEQ.ID.NO.57)
TYR 171: AATATTTATGACCTCTTTGTCTGGATG (SEQ ID NO:58)
TYR 444: GATCTGGGCTATGACTATAGCTATCTA (SEQ ID NO:59)
TYR 57: AATATCCTTCTGTCCAATGCACCACTT (SEQ ID NO:60)
TRP-1 245: TCCCTTCCTTACTGGAATTTTGCAACG (SEQ ID NO:61)
TRP-1 298: ACCCTGGGAACACTTTGTAACAGCACC (SEQ ID NO:62)
TRP-1 481: ATAGCAGTAGTTGGCGCTTTGTTACTG (SEQ ID NO:63)
TRP-1 181: AACATTTCCATTTATAACTACTTTGTT (SEQ IDNO:64)
TRP-1 439: AACATGGTGCCATTCTGGCCCCCAGTC (SEQ ID NO:65)
hPER1-1-gp100(280-288)
AGC AGG AGG CAC CAC TGC AGG AGC AAG GCC AAG AGG AGC AGG
CAC
CAC TAC CTG GAG CCC GGC CCC GTG ACC GTG (SEQ ID NO:66)
hPER1-2-gp100(154-162)
AGG AGG CAC CAC AGG AGG AGC AAG GCC AAG AGG AGC AGG AAG
ACC TGG GGC CAG TAC TGG CAG GTG (SEQ ID NO:67)
hPER1-2-F-gp100(154-162)
AGG AGG CAC CAC AGG AGG AGC AAG GCC AAG AGG AGC AGG TTC
GTG TAC GTG TGG AAG ACC TGG GGC CAG TAC TGG CAG GTG (SEQID NO:68)
Claims (21)
1.多肽,其基本上由连接到包含细胞毒性T淋巴细胞表位的第二个氨基酸序列的包含hPER1的转导序列的第一个氨基酸序列组成,其中所述转导序列是RRHHRRSKAKRSR。
2.权利要求1的多肽,其中在第一个和第二个氨基酸序列之间插入接头序列。
3.权利要求2的多肽,其中所述接头序列与第二个氨基酸序列天然发生。
4.权利要求2的多肽,其中所述接头序列不与第二个氨基酸序列天然发生。
5.权利要求1的多肽,其中所述第二个氨基酸序列来自肿瘤抗原、传染物的抗原或者自身免疫抗原。
6.组合物,其包含可药用载体中的权利要求1-5任一项的多肽。
7.免疫宿主的方法,其包括对该宿主施用权利要求6的组合物。
8.免疫宿主的方法,其包括将权利要求1-7任一项的多肽或者组合物与树突细胞混合以产生负荷肽的树突细胞并对宿主施用该负荷肽的树突细胞。
9.分离的重组DNA分子,其包含与编码hPER1的转导序列的第二个DNA序列连接的编码细胞毒性T淋巴细胞表位的第一个DNA序列,其中所述转导序列是RRHHRRSKAKRSR。
10.权利要求21的DNA分子,其中将编码接头氨基酸序列的DNA序列插入第一个和第二个氨基酸序列之间。
11.权利要求22的DNA分子,其中所述接头氨基酸序列与第二个氨基酸序列天然发生。
12.权利要求11的DNA分子,其中所述接头序列不与第二个氨基酸序列天然发生。
13.权利要求9-12任一项的DNA分子,其中所述第一个氨基酸序列来自肿瘤抗原、传染物的抗原或者自身免疫抗原。
14.组合物,其包含权利要求9-14任一项的重组DNA分子。
15.免疫宿主的方法,其包括通过皮下、皮内或者鼻内途径施用基本上由第一个氨基酸序列组成的多肽,所述多肽包含施用的权利要求1-14任一项的多肽、重组DNA或者组合物。
16.权利要求16的方法,其中细胞毒性T淋巴细胞表位来自肿瘤抗原、传染物或者自身免疫抗原。
17.免疫宿主的方法,其包括通过皮下、皮内或者鼻内途径施用基本上由权利要求1-14任一项的多肽、重组DNA或者组合物组成的靶定的免疫原。
18.免疫宿主的方法,其包括通过皮下、皮内或者鼻内途径施用靶定的免疫原,该免疫原基本上由连接到包含细胞毒性T淋巴细胞表位的第二个氨基酸序列的包含hPER1的转导序列的多肽组成。
19.免疫宿主的方法,其包括通过皮下、皮内或者鼻内途径施用靶定的免疫原,该免疫原基本上由包含连接到编码hPER1的转导序列的第二个DNA序列的重组DNA的编码细胞毒性T淋巴细胞表位的第一个DNA序列的重组DNA分子组成。
20.免疫宿主的方法,其包括通过皮下、皮内或者鼻内途径施用包含权利要求18的多肽或者权利要求19的重组DNA分子的组合物。
21.权利要求17-20任一项的方法,其中所述细胞毒性T淋巴细胞表位来自肿瘤抗原、传染物或者自身免疫抗原。
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WO2016131856A1 (en) * | 2015-02-18 | 2016-08-25 | F. Hoffmann-La Roche Ag | Immunoconjugates for specific induction of t cell cytotoxicity against a target cell |
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JP2007536911A (ja) | 2007-12-20 |
EP1699492A2 (en) | 2006-09-13 |
WO2005066203A2 (en) | 2005-07-21 |
BRPI0418273A (pt) | 2007-05-02 |
IL176603A0 (en) | 2006-10-31 |
ZA200605304B (en) | 2007-12-27 |
US20060002946A1 (en) | 2006-01-05 |
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