CN101282742B - 脂质体在含连续疏水相的载体中作为癌症治疗媒介物的应用 - Google Patents
脂质体在含连续疏水相的载体中作为癌症治疗媒介物的应用 Download PDFInfo
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Abstract
本发明公开了一种组合物,其包含连续疏水相和作为输送能够诱导细胞毒性T淋巴细胞(CTL)反应的抗原的媒介物的脂质体,并公开了所述组合物在癌症治疗中的应用方法。
Description
相关申请的交叉引用
本申请要求分别在2005年10月7日、2006年1月13日和2006年4月7日递交的加拿大专利申请No.2,523,032;2,533,705和2,542,212的权益和优先权,并进一步要求2006年7月5日递交的美国临时专利申请No.60/806,573的权益和优先权,所有申请均经由引用而合并于此。
技术领域
本申请涉及一种包含脂质体和连续疏水相的组合物在癌症治疗中的应用,所述组合物作为用于输送能够诱导细胞毒性T淋巴细胞(CTL)反应的抗原的媒介物。
背景技术
以前在已有技术中已经记载了包含脂质体和各种抗原的长效疫苗。这些疫苗组合物在诱导依赖于T辅助细胞2(Th2)的功能的针对特定抗原的增强的体液免疫反应(通过增加的抗体产量测定)方面表现出有效性。但是,对于一种破坏癌症的组合物来说,其必需能够诱导细胞介导(细胞毒性T淋巴细胞(CTL))的反应。CTL是T淋巴细胞的一个亚群,其能够诱导感染的体细胞或肿瘤细胞死亡;它们杀死(溶解)被病毒(或其他病原体)感染的或者被破坏或功能失调的细胞。CTL反应通过T辅助细胞1(Th1)细胞因子介导。
总的来说,CTL反应是短期的,仅仅持续几个星期(Knutson et al.,Clin.Cancer.Res.8(5):1014-1018,1990;Dudley et al.,J.Immunother.24(4):363-73,2002;and Fernando et al.,Scand.J.Immunol.47(5):459-65,1998)。而癌症的 复发总是人们关注的问题,所以长效CTL反应的诱导对于保证癌症不复发是必需的。
所以,仍需要开发不需多次加强剂量治疗的用于癌症治疗的长效免疫治疗组合物。
发明内容
在一个实施方式中,提供了一种组合物,包括:含有连续的疏水物质相的载体;脂质体;和能够诱导CD8+细胞毒性T淋巴细胞(CTL)反应的至少一种抗原。优选这种组合物还包括至少一种T辅助细胞表位。
本申请的进一步的方面提供了一种在被治疗者中治疗癌症的方法,包括以此处所述的组合物给药。
根据另一个方面,本申请提供了用于在被治疗者中治疗癌症的试剂盒,包括此处所述的组合物以及其使用说明书。
通过下面结合附图的本发明的具体实施方式的描述,对于本领域技术人员来说本发明的其他方面和特征将变得明显。
附图说明
这些附图仅仅以示例的方式说明本发明的实施方式:
图1说明了来自小鼠的脾细胞的离体胞内IFN-γ染色,所述小鼠在用组合物处理14天后,分别暴露于HPV16的E7表位(R9F肽;SEQ ID NO:1)、或无关肽中、或无肽环境中,所述组合物含有在PBS/FIA油包水乳液中含有的脂质体中的胶囊化的CpG寡聚脱氧核苷酸(CpG ODN)(SEQ ID NO:12)和融合了PADRE的R9F肽。处理后暴露于R9F时,没有一个对照处理引起高于背景水平的CD8+/IFN-γT细胞的扩增(数据未示出)。相反,比起暴露于无关肽的小鼠,暴露于R9F肽的小鼠的脾细胞中产生的R9F-反应性CTL升高了12.9倍。
图2说明了在30天前事先用组合物处理的小鼠的脾细胞对载以R9F肽 (SEQ ID NO:1)(方块)和无关肽(菱形)的EL-4细胞的裂解,所述组合物含有融合了PADRE的R9F肽(SEQ ID NO:10)与CpG ODN(SEQ ID NO:12)一起在悬浮于PBS/FIA油包水乳液中的脂质体中胶囊化。
图3说明了用下列组合物之一处理过的小鼠脾细胞对EL-4细胞的裂解:(i)在悬浮于PBS/FIA油包水乳液中的脂质体中胶囊化的融合肽(融合以PADRE(SEQ ID NO:10)的R9F肽(SEQ ID NO:1))和CpG ODN(SEQ ID NO:12)(菱形);(ii)在PBS/FIA油包水乳液中未胶囊化的融合肽和CpG ODN(SEQ ID NO:12)(三角形和叉形);或(iii)在PBS/FIA油包水乳液中胶囊化融合肽的脂质体(实心圆圈)。然后,在处理后130天将脾细胞或者暴露于R9F肽(叉形、菱形和实心圆圈)或暴露于无关肽(SEQ ID NO:13)(三角形和空心圆圈)。只用融合肽处理的小鼠脾细胞在处理后130天暴露于R9F或无关肽的EL-4细胞的裂解显示为基准水平(数据未示出)。
图4说明了用含有悬浮在PBS/FIA油包水乳液中的脂质体中胶囊化的CpG ODN、融合肽(R9F肽(SEQ ID NO:1)与PADRE(SEQ ID NO:10)融合)的组合物单次给药处理(空心方块)对小鼠中的14天的C3肿瘤大小的影响。到第30天(处理后16天)肿瘤用触诊的方法探查不到。相反,在用不含融合肽的上述组合物(实心方块)对照处理的小鼠中,C3肿瘤继续长大。所有小鼠(n=10)在处理前14天用0.5×106个C3肿瘤细胞攻击。两个处理组中的肿瘤大小的差异有统计学意义(在第25天p=0.002)。
图5说明了对用0.5×106个C3细胞攻击之后61天没有发生肿瘤的小鼠的百分比方面的预防处理效果。处理组由用含有下列物质的组合物之一处理的小鼠组成:(1)PBS;(2)CpG于PBS中;(3)融合肽(与PADRE(SEQ IDNO:10)融合的R9F肽(SEQ ID NO:1))于PBS中;(4)在悬浮于PBS/FIA油包水乳液中的脂质体中胶囊化的融合肽;(5)油包水乳液中的融合肽和CpGODN;(6)在悬浮在油包水乳剂中的脂质体中胶囊化的融合肽和CpG ODN;(7)在悬浮于油包水乳液中的脂质体中胶囊化的融合肽和脂肽Pam3Cys-SKKKK(Pam3c)。
图6说明了用下述组合物之一单次处理之后8天的小鼠中TRP-2特异性产IFN-γ脾细胞(斑点形成细胞,SFC)的离体检测:(A)含有在悬浮于PBS/ISA51油包水乳液中的脂质体中胶囊化的带有PADRE(SEQ ID NO:10)的酪氨酸酶相关蛋白-2(TRP-2)肽(S9L;SEQ ID NO:7)和CpG ODN(SEQ IDNO:12)的组合物;(B)含有在悬浮于油包水乳液中的脂质体中的带有PADRE的S9L的组合物;或(C)含有在悬浮于PBS/ISA51油包水乳液中的脂质体中的带有CpG的无关肽(SEQ ID NO:13)的组合物。
图7说明了用组合物(A)单次处理之后的小鼠中的p53特异性产IFN-γ脾细胞(SFC)的离体检测,组合物(A)含有悬浮于PBS/ISA51油包水乳液中的脂质体胶囊化的带有PADRE(SEQ ID NO:10)的修饰的p53肽(mK9M;SEQ ID NO:8)和CpG ODN(SEQ ID NO:12)的组合物;或下列对照组合物之一单次处理之后的小鼠中的p53特异性产IFN-γ脾细胞(SFC)的离体检测:(B)在油包水乳液中的带有PADRE的mK9M;(C)带有PADRE的mK9M在脂质体中胶囊化并悬浮于油包水乳液中;和(D)带有CpG ODN的无关肽(SEQ ID NO:13)在脂质体中胶囊化并悬浮于油包水乳液中。
图8说明了用组合物(A)单次处理之后的小鼠中的TRP-2和p53-特异性产IFN-γ的脾细胞(SFC)的离体检测,所述组合物(A)包括在悬浮于PBS/ISA51油包水乳液中的脂质体中胶囊化的带有PADRE(SEQ ID NO:10)的p53(mK9M肽;SEQ ID NO:8)和TRP-2(V8L;SEQ ID NO:6)肽和CpGODN(SEQ ID NO:12);或用下列对照组合物之一单次处理之后的小鼠中的TRP-2和p53-特异性产IFN-γ的脾细胞(SFC)的离体检测:(B)的带有PADRE的p53和TRP-2肽在脂质体中胶囊化并悬浮于PBS/ISA51油包水乳液中;(C)带有PADRE的p53和TRP-2肽和CpG ODN;(D)带有PADRE的p53和TRP-2肽。
图9说明了用含有HLA-A2 E6/7CTL表位的肽在已建立的19日龄TC1/A2肿瘤上单次处理的效果。用下述组合物单次处理后,到第21天时消 除了TC1/A2肿瘤,所述组合物包含四个HPV E6/7肽(Y10T(SEQ ID NO:2)、L9V(SEQ ID NO:3)、T81(SEQ ID NO:4)和T10V(SEQ ID NO:5))的混合物与CpG ODN(SEQ ID NO:12)和PADRE(SEQ ID NO:10)在脂质体中胶囊化然后悬浮于PBS/ISA51油包水乳液中(方块);或者所述组合物包括一个用“aay”(丙氨酸-丙氨酸-酪氨酸)连接体将上述四个HPV E6/7肽连接到一起的长肽(AB2肽;SEQ ID NO:14)与CpG ODN和PADRE在脂质体中胶囊化然后悬浮于油包水乳液中(菱形)。用包含一个HPV的E7肽(L9V;SEQ ID NO:3)并与CpG ODN和PADRE在脂质体中胶囊化然后悬浮于油包水乳液中的组合物的处理显著减小了肿瘤的大小(三角形)。仅用PBS的处理(叉形)不能防止肿瘤的生长。
图10说明在肿瘤移植后19天仅用PBS注射的5只小鼠的肿瘤生长。
图11说明在五只小鼠中,用下述组合物单次处理对已建立的19日龄TC1/A2肿瘤的生长的作用,所述组合物含有四个含HLA-A2 E6/7CTL表位的肽(Y10T、L9V、T81和T10V;SEQ ID NOs:2,3,4和5)的混合物与CpGODN(SEQ ID NO:12)和PADRE(SEQ ID NO:10)在脂质体中胶囊化然后悬浮于PBS/ISA51油包水乳液中。
图12说明了在5只小鼠中用含有一个长肽(AB2;SEQ ID NO:14)与CpGODN(SEQ ID NO:12)和PADRE(SEQ ID NO:10)在脂质体中胶囊化然后悬浮于PBS/ISA51油包水乳液中的组合物单次处理对已建立的19日龄的TC1/A2肿瘤的生长的影响。
图13说明了在5只小鼠中用含有HLA-A2 E7 CTL表位(L9V;SEQ IDNO:3)与CpG ODN(SEQ ID NO:12)和PADRE(SEQ ID NO:10)在脂质体中胶囊化然后悬浮于PBS/ISA51油包水乳液中的组合物单次处理对已建立的19日龄的TC1/A2肿瘤的生长的影响。
图14说明了在用含有(A)或(B)的组合物单次处理免疫后9天的小鼠中产IFN-γ的脾细胞的离体检测,其中(A)为四个短的未连接的含HPV-A2 HPV E6/E7 CTL表位的肽(T81、Y10T、L9V或T10V;SEQ ID Nos分别为:2,3,4和5);(B)为用“kkp”连接体连接到一起的两个中长的二肽(Y10T-kkp-L9V(SEQ ID NO:15)和T81-kkp-T10V(SEQ ID NO:16))。这两种组合物都是与CpG ODN(SEQ ID NO:12)和PADRE(SEQ ID NO:10)在脂质体中胶囊化然后悬浮于PBS/ISA51油包水乳液中。来自对照小鼠(C)的脾含有背景数量的能够用每个单独的短肽或四个短肽的混合物刺激产生IFN-γ的脾细胞。相反,在用四个未连接的短肽或两个中长的二肽免疫的组中的小鼠的脾含有更多数量的被这些肽刺激产生IFN-γ的脾细胞。用本发明的两个配方中的任何一个进行免疫并用四个短肽的混合物刺激的小鼠的脾细胞的刺激产生的产IFNγ脾细胞大约是对照脾细胞的五倍,说明用“kkp”连接的肽对免疫反应没有作用。
图15说明在用下列组合物之一处理的小鼠中的黑色素瘤的生长,这些组合物含有与CpG ODN和PADRE在脂质体中胶囊化并悬浮于油包水乳液中的至少一种源自黑色素瘤相关蛋白(TRP-2或p53)的肽,所述组合物为:(i)V8L肽(SEQ ID NO:6)(菱形);(ii)S9L肽(SEQ ID NO:7)(方块);(iii)mK9M肽(SEQ ID NO:8)(三角形);(iv)V8L和mK9M肽(叉形);和(v)S9L和mK9M肽(星形)。对照小鼠仅用PBS处理(圆圈)。肿瘤移植后5天,对小鼠进行所述处理。每个数据点都是五只小鼠的肿瘤的平均大小。
图16说明说明在用下列组合物之一处理后的带有黑色素瘤的小鼠的百分率,这些组合物含有与CpG ODN(SEQ ID NO:12)和PADRE(SEQ ID NO:10)在脂质体中胶囊化并悬浮于PBS/ISA51油包水乳液中的至少一种源自黑色素瘤相关蛋白(TRP-2或p53)的肽,所述组合物为:(i)V8L肽(SEQ ID NO:6)(菱形);(ii)S9L肽(SEQ ID NO:7)(方块);(iii)mK9M肽(SEQ ID NO:8)(三角形);(iv)V8L和mK9M肽(叉形);和(v)S9L和mK9M肽(星形)。对照小鼠仅用PBS处理(圆圈)。肿瘤移植后5天,对小鼠进行所述处理。每个数据点都是五只小鼠中的肿瘤的平均大小。
图17说明用含有CpG ODN(SEQ ID NO:12)、PADRE和TRP-2和/或p53CTL表位的在悬浮于PBS中然后在ISA51中乳化的脂质体中胶囊化的组合物单次处理后,对小鼠的已建立的6日龄B16肿瘤的消除或减小。使用TRP-2和p53CTL表位混合物的单次处理使得给药后21天所有的小鼠都消除了肿瘤(三角形)。只用TRP-2(菱形)或p53(方块)单次处理使得40%的小鼠在处理后33天后消除了肿瘤。在对照小鼠中用PBS处理对肿瘤的发展没有作用(叉形)。
图18说明在用包含在脂质体中胶囊化并悬浮在PBS/ISA51油包水乳液中的CpG ODN(SEQ ID NO:12)和HPV 16的E7表位(R9F肽;SEQ ID NO:1)的组合物处理,然后在15~20小时内在给药位点用AldaraTM乳膏(5%咪喹莫特)进行皮肤涂敷(方形)的小鼠中,C3肿瘤的尺寸减小了。相反,C3肿瘤在接受PBS处理(叉形)或PBS处理之后15~20小时内在PBS注射位点进行Aldara皮肤涂敷(三角形)的对照组中没有减小。在所有的处理组中,所述处理为肿瘤移植5天后进行给药。肿瘤大小为10只小鼠中肿瘤的平均大小。
图19说明了在所有的处理组中,肿瘤移植5天后进行处理,处理之后具有明显肿瘤的小鼠的百分率。肿瘤移植后20天,仅有20%的小鼠在用包含在脂质体中胶囊化并悬浮在PBS/ISA51油包水乳液中的CpG ODN(SEQID NO:12)和HPV 16的E7表位(R9F肽;SEQ ID NO:1)的组合物处理,然后在15~20小时内在给药位点用AldaraTM乳膏(5%咪喹莫特)进行皮肤涂敷(菱形)后具有明显的肿瘤。相反,单纯用PBS处理(三角形)后20天90%的小鼠具有明显的肿瘤,用PBS处理后皮肤涂敷Aldara(方块)处理之后的100%的小鼠具有明显的肿瘤。
图20说明了用包含S9L肽和破伤风类毒素表位(F21E;SEQ ID NO:11)与CpG ODN(SEQ ID NO:12)一起在PBS/ISA51油包水乳液中的脂质体中胶囊化的处理组合物(B)单次给药免疫后8天,暴露于黑色素瘤相关抗原、 TRP-2(S9L肽;SEQ ID NO:7)或B16F10细胞的产IFN-γ的脾细胞的离体检测(B)。对照小鼠用配方中没有破伤风类毒素T辅助细胞表位的上述组合物(A)免疫。用本发明的处理组合物免疫的组的脾中存在的用B16-F10细胞刺激产生IFN-γ的脾细胞是对照组的5倍。
具体实施方式
本申请提供一种组合物,其包括至少一种能够诱导CD8+细胞毒性T淋巴细胞(CTL)反应的抗原,与至少一个T辅助细胞表位以及脂质体一起悬浮在包含连续的疏水物质相的载体中。本发明进一步说明了所述组合物在被治疗者中用于治疗癌症的方法中的应用。
此处描述的组合物用于治疗广泛范围的癌症,包括,但不限于:由人乳头状瘤病毒(HPV)引起的癌症,举例来说,如宫颈癌和/或外阴癌;涉及酪氨酸酶的表达的癌症,举例来说,如黑色素瘤;涉及p53基因产物突变或过表达的癌症,举例来说,如乳腺癌或淋巴结转移瘤;和其他癌症,比如同时表达多于一种肿瘤相关蛋白的黑色素瘤。在另一个实施方式中,所述组合物用于治疗包括但不限于如下的癌症:肺癌、卵巢癌、多发性骨髓瘤、B细胞淋巴瘤、肝细胞瘤、肉瘤、膀胱癌、前列腺癌、甲状腺肿瘤、头颈肿瘤、结肠癌、直肠癌、肾癌、胰腺癌、胃癌、腺癌、T细胞白血病、淋巴肉瘤、子宫肿瘤、食道肿瘤、非何杰金氏淋巴瘤、子宫内膜瘤、和RCC(肾细胞癌)肿瘤。具有与所述癌症源自的细胞类型的细胞表面成分的数量或本质不同的任何癌症都是本发明治疗的候选者。具体来讲,p53是广泛用于癌症治疗的候选靶点(DeLeo,A.B.,Crit.Rev.Immunol.,18:29,1998;Vierboom,M.P.M.etal.,Peptide-Based Cancer Vaccines.W.M.Kast,ed.Landes Bioscience,Georgetown,2000)。
此处使用的术语“肿瘤”、“肿瘤细胞”、“癌症”和“癌症细胞”(可互换使用)指那些呈现不正常的生长,以显著丧失对细胞增殖的控制为特征 的细胞或那些已经永生的细胞。术语“癌症”或“肿瘤”包括转移性的和非转移性的癌症或肿瘤。癌症,包括恶性肿瘤的存在可以用本领域普遍接收的标准诊断。
“治疗”癌症指一种得到有益的或想要的结果的方法,包括临床结果。有益的或想要的临床结果可包括但不限于,一种或多种症状或不适的缓和或改善、疾病范围程度的减小、疾病状态的稳定、疾病发展的预防、疾病扩散的预防、疾病发展升级的延迟或减缓、疾病发作的延迟或减缓、疾病状态的改善与减轻、以及缓解(无论是部分还是全部)。“治疗”也可指与不治疗的预期相比延长生存期。“治疗”也可指临时抑制疾病的发展升级,虽然更优选的是其涉及在被治疗者中使疾病的发展永久地停止。
要治疗的被治疗者可以是任何脊椎动物,优选哺乳动物,更优选是人。抗原
所述组合物的适宜抗原是那些能够在被治疗者体内诱导细胞介导的(CTL)免疫反应的抗原。
细胞介导的免疫是一种不涉及抗体,而涉及巨噬细胞和NK细胞的激活、抗原特异的细胞毒性T淋巴细胞产生和响应于抗原的各种细胞因子的释放的免疫反应。细胞毒性T淋巴细胞是T淋巴细胞(白细胞的一种)的亚群,能够诱导感染的体细胞或肿瘤细胞死亡,它们杀死感染了病毒(或其他病原体)或者要不然被破坏或机能障碍的细胞。
大多数细胞毒性T细胞表达能识别结合到I类MHC分子上的特异肽抗原的T细胞受体。这些CTL还表达与I类MHC分子的部位亲和的CD8(CD8+ T细胞)。这种亲和性保持了这些CTL和靶细胞在抗原特异性激活中的紧密结合。
细胞免疫通过诸如如下方式来保护机体,例如激活抗原特异性细胞毒性T淋巴细胞,其能够裂解在其表面呈现外来抗原表位的体细胞,例如病毒感染的细胞、带有胞内细菌的细胞和呈现肿瘤抗原的癌细胞;激活巨噬细胞和 NK细胞,使其破坏细胞内病原体;和刺激细胞分泌各种细胞因子,这些细胞因子影响涉及适应性免疫反应和固有免疫反应的其他细胞的功能。
在一个实施方式中,所述抗原可以是例如,能够在被治疗者中产生CTL免疫反应的肽、适宜的固有的、非固有的、重组或变性蛋白或多肽、或其片段、或表位。
术语“多肽”包括氨基酸的任何链而不考虑长度(例如至少6、8、10、12、14、16、18或20个氨基酸),或翻译后修饰(例如糖基化或磷酸化),并包括,例如天然蛋白、合成或重组多肽和肽、表位、杂交分子、变体、同系物、类似物、类肽、素拟肽等。因此变体或衍生物包括缺失,所述缺失包括平截和片段;插入和添加,例如保守替换、定点突变和等位基因变体;和修饰,包括具有共价连接到所述肽的一个或多个非氨基酰基基团(例如糖、脂等)的类肽和翻译后修饰。此处使用的术语“保守氨基酸替换”或“保守替换”指在肽中给定的位点由一个氨基酸替换另一个,其中进行替换而相关功能基本不丧失。在进行这种改变时,相似氨基酸残基的取代可以基于侧链取代基的相关相似性进行,例如它们的大小、电荷、疏水性、亲水性等,并且这样的取代可以用常规检测来分析其对肽功能的影响。
可以使用与此处公开的基本相同的多肽、肽或表位。如果当经过最优对齐(允许有间隙)它们具有至少大约50%的序列一致性,或者如果这些序列共有限定的功能模体的话,这两个序列就被认为是具有基本一致性。在可替代的实施方式中,如果最优对齐的序列在一特定区域其具有至少60%、70%、75%、80%、85%、90%、95%、96%、97%、98%、99%的一致性,则被认为是基本相同的(即具有基本一致性)。术语“一致性”指的是两个多肽分子间的序列相似性。一致性可以通过在对齐的序列中比较每个位点来确定。氨基酸序列间的一致性程度是这些序列,例如特定区域中共有的位点处相同或匹配的氨基酸数量的函数。用于一致性比较的序列的最优对齐可以用本领域已知的各种算法来进行,包括:ClustalW程序,可从 http://clustalw.genome.ad.jp得到;局部同源性算法(Smith和Waterman,1981,Adv.Appl.Math 2:482);同源性对齐算法(Needleman和Wunsch,1970,J.Mol.Biol.48:443);相似性搜索方法(Pearson和Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444);和这些算法的计算机化工具(例如美国成斯康星州麦迪逊遗传学计算机集团的威斯康星遗传学软件包中的GAP、BESTFIT、FASTA和TFASTA)。序列一致性也可以用BLAST算法来确定,该算法记载于Altschul et al.,1990,J.Mol.Biol.215:403-10(采用公开的默认设置)。进行BLAST分析的软件可以从生物技术信息国家中心得到(从互联网 http://www.ncbi.nlm.nih.gov/得到)。
用在此所述的组合物的单独治疗中使用的抗原的量可以根据抗原的类型和被治疗者的身形尺寸而改变。本领域技术人员不需过度实验即能确定在具体应用中需用的抗原的有效量。此处使用的术语“有效量”意为在所需的剂量和时间段内为达到理想结果的有效的量。
在一个实施方式中,所述抗原可以是能够诱导CTL反应的至少一种CTL表位。例如,所述抗原可以是来源于诸如HPV的病毒的CTL表位。
在另一个实施方式中,所述抗原可以是选自由来源于HPV的E6或E7蛋白的表位所组成的组中的CTL表位。
在进一步的实施方式中,HPV的E6蛋白的表位包含肽序列TIHDIILECV(T10V)(SEQ ID NO:5)。在另一个实施方式中,HPV的E7蛋白的表位包含选自由下列序列组成的组中的肽序列:RAHYNIVTF(R9F)(SEQ ID NO:1)、YMLDLQPETT(Y10T)(SEQ ID NO:2)、LLMGTLGIV(L9V)(SEQ ID NO:3)和TLGIVCPI(T81)(SEQ ID NO:4)。
在另一个实施方式中,所述CTL表位可以是肿瘤相关蛋白的表位,举例来说,如黑色素瘤相关蛋白。在进一步的实施方式中,所述黑色素瘤相关蛋白是酪氨酸相关蛋白-2(TRP-2)或p53,其能够通过包括重组技术或化学合成的各种方法得到。
在一个实施方式中,来源于TRP-2的蛋白的表位包含例如SVYDFFVWL(S9L;SEQ ID NO:7)的肽序列。在另一个实施方式中,来源于TRP-2的蛋白的表位包含肽序列VYDFFVWL(V8L;SEQ ID NO:6)。在另一个实施方式中,p53来源的蛋白的表位包含选自KYMCNSSCM(K9M;野生型p53;SEQID NO:9)、KYICNSSCM(mK9M;修饰的p53;SEQ ID NO:8)和AKXVAAWTLKAAAKYICNSSCM(mK9M(SEQ ID NO:9)与PADRE(SEQID NO:10)连接)的肽序列。
在一个实施方式中,所述组合物可包括CTL表位的混合物作为诱导CTL反应的抗原。
在进一步的实施方式中,所述抗原可以是能够诱导能有效识别所针对的肿瘤细胞上的特定构造并引起其破坏的特异性CTL反应的任何肽或多肽。
在再进一步的实施方式中,所述抗原可包括选自下表的肽序列:
表1
抗原 | 序列 | HLA | 专利 |
Mart-1/ Melan-A | AAGIGILTV (SEQ ID NO:18) | A2 | US 5,844,075 |
EAAGIGILTV (SEQ ID NO:19) | A2 | US 5,844,075 | |
ILTVILGVL (SEQ ID NO:20) | A2 | US 5,844,075 | |
AEEAAGIGIL (SEQ ID NO:21) | B45 | US 7,037,509 | |
AEEAAGIGILT (SEQ ID NO:22) | B45 | 未知 | |
MCIR | TILLGIFFL (SEQ ID NO:23) | A2 | 未知 |
FLALIICNA (SEQ ID NO:24) | A2 | 未知 | |
Gp100 | KTWGQYWQV (SEQ ID NO:25) | A2 | US 5,844,075 |
AMLGTHTMEV (SEQ ID NO:26) | A2 | 未知 | |
MLGTHTMEV (SEQ ID NO:27) | A2 | 未知 |
[0055]
抗原 | 序列 | HLA | 专利 |
Gp100 | SLADTNSLAV (SEQ ID NO:28) | A2 | US 5,844,075 |
ITDQVPFSV (SEQ ID NO:29) | A2 | US 5,844,075 | |
LLDGTATLRL (SEQ ID NO:30) | A2 | US 5,844,075 | |
YLEPGPVTA (SEQ ID NO:31) | A2 | US 5,844,075 | |
RLPRIFCSC (SEQ ID NO:33) | A2 | 未知 | |
LIYRRRLMK (SEQ ID NO:34) | A3 | 未知 | |
ALNFPGSQK (SEQ ID NO:35) | A3 | 未知 | |
SLIYRRRLMK (SEQ ID NO:36) | A3 | 未知 | |
ALLAVGATK (SEQ ID NO:37) | A3 | US 6,558,671 | |
ALLAVGATK (SEQ ID NO:38) | A3 | US 6,977,074 | |
VYFFLPDHL (SEQ ID NO:39) | A24 | 未知 | |
SNDGPTLI (SEQ ID NO:40) | Cw8 | 未知 | |
PSA | VSHSFPHPLY (SEQ ID NO:41) | A1 | US 6,037,135 |
FLTPKKLQCV (SEQ ID NO:42) | A2 | US 6,881,405 | |
VISNDVCAQV (SEQ ID NO:43) | A2 | 未知 | |
PSM | HSTNGVTRIY (SEQ ID NO:44) | A1 | 未知 |
酪氨酸酶 | KCDICTDEY (SEQ ID NO:45) | A1 | US 7,019,112 |
SSDYVIPIGTY (SEQ ID NO:46) | A1 | 未知 | |
YMDGTMSQV (SEQ ID NO:47) | A2 | US 6,096,313 | |
MLLAVIYCL (SEQ ID NO:48) | A2 | US 6,291,430 | |
AFLPWHRLF (SEQ ID NO:49) | A24 | US 6,291,430 | |
SEIWRDIDF (SEQ ID NO:50) | B44 | US 6,291,430 | |
MSLQRQFLR (SEQ ID NO:51) | A31 | US 5,831,016 |
[0056]
抗原 | 序列 | HLA | 专利 |
TRP1 | SVYDFFVWL (SEQ ID NO:52) | A2 | US 7,067,120 |
TRP2 | TLDSQVMSL (SEQ ID NO:53) | A2 | 未知 |
LLGPGRPYR (SEQ ID NO:54) | A31 | US 5,831,016 | |
p53 | ANDPIFVVL (SEQ ID NO:55) | Cw8 | 未知 |
如上表1所示,蛋白质(多肽)在可作为CTL表位并因此能用于本发明的肽序列的数量上有所变化。下列基因,但不限于此,编码具有可以作为抗原合并入本发明的肽序列的肿瘤相关蛋白:p53、HPV E6和E7、ART-4、CAMEL、CEA、Cyp-B、HER2/neu、hTERT、hTRT、iCE、MUC1、MUC2、PRAME、P15、RUI、RU2、SART-1、SART-3、WT1、PSA、酪氨酸酶、TRP-1、TRP-2、gp100、MART-1/Melan A、MAGE-A1、MAGE-A2、MAGE-A3、MAGE-A6、MAGE-A10、MAGE-A12、BAGE、DAM-6、DAM-10、GAGE-1、GAGE-2、GAGE-3、GAGE-4、GAGE-5、GAGE-6、GAGE-7B、GAGE-8、NA88-A、NY-ESO-1、NY-ESO-1a(CAG-3)、AFP、β-连环蛋白/m、天冬氨酸特异性半胱氨酸蛋白酶-8/m、CDK-4/m、ELF2M、GnT-V、G250、Ras、HSP70-2M、HST-2、KIAA0205、MUM-1、MUM-2、MUM-3、肌球蛋白/m、RAGE、SART-2、TRP-2/INT2和707-AP。
T辅助细胞表位
T辅助细胞表位是具有T辅助细胞活性的氨基酸序列(天然或非天然氨基酸)。T辅助细胞表位被T辅助细胞淋巴细胞识别,这在建立和最大化免疫系统能力方面行使重要功能,并参与激活和指导其他免疫细胞,例如细胞毒性T淋巴细胞。
T辅助细胞表位可由连续的或不连续的表位组成。所以不是T辅助细胞的每个氨基酸都是所述表位的必需部分。所以,T辅助细胞表位,包括T辅助细胞表位的类似物和片段,能够增强或刺激免疫反应。免疫显性的T辅助 细胞表位在具有广泛趋异的MHC型的动物和人群中普遍有活性(Celis et al.(1988)J.Immunol.140:1808-1815;Demotz et al.(1989)J.Immunol.142:394-402;Chong et al.(1992)Infect.Immun.60:4640-4647)。目标肽的T辅助细胞结构域具有约10~约50个氨基酸,优选约10~约30个氨基酸。当存在多个T辅助细胞表位时,每个T辅助细胞表位都独立作用。
在一个实施方式中,此处描述的组合物还包含至少一个T辅助细胞表位。在某些情况下,T辅助细胞表位可构成所述抗原的一部分。特别是,如果所述抗原具有足够的大小,它可以含有行使T辅助细胞表位功能的表位。在其他实施方式中,所述T辅助细胞表位是独立于所述抗原的单独的分子。
在另一个实施方式中,T辅助细胞表位类似物可包括在T辅助细胞表位中置换、缺失和插入1个至约10个氨基酸残基。T辅助细胞片段是足以增强或刺激免疫反应的T辅助细胞表位的毗连部分。T辅助细胞片段的一个例子是来源自单个较长的肽的一系列重叠的肽。
用于本发明的T辅助细胞表位的来源包括,例如,乙型肝炎表面抗原辅助T细胞表位、百日咳毒素辅助T细胞表位、麻疹病毒F蛋白辅助T细胞表位、衣原体Chlamydia trachomitis主要外膜蛋白辅助T细胞表位、白喉毒素辅助T细胞表位、恶性疟原虫(Plasmodium falciparum)环子孢子辅助T细胞表位、曼森血吸虫(Schistosoma mansoni)磷酸丙糖异构酶辅助T细胞表位、大肠杆菌TraT辅助T细胞表位和这些T辅助细胞表位的免疫增强类似物和片段。
在一个实施方式中,T辅助细胞表位是通用的T辅助细胞表位。此处使用的通用T辅助细胞表位指以II类(CD4+T细胞)或I类(CD8+T细胞)限制性模式激活T细胞功能的方式结合到多种MHC II类分子的肽或其他免疫原性分子或其片段。
在另一个实施方式中,所述T辅助细胞表位可以是诸如PADRE(pan-DR表位)的通用T辅助细胞表位,包含肽序列AKXVAAWTLKAAA(SEQ ID NO: 10),其中X可以是环己基丙氨酰。PADRE特别具有CD4+T-辅助细胞表位,即,它刺激PADRE-特异的CD4+T辅助细胞反应的诱导。
破伤风类毒素具有以与PADRE相似的方式行使功能的T辅助细胞表位。破伤风毒素和白喉毒素具有对于人类CD4+细胞的通用表位(Diethelm-Okita,B.M.et al.,Universal表位for human CD4+cells on tetanusand diphtheria toxins.J.Infect.Diseases,181:1001-1009,2000)。在另一个实施方式中,所述T辅助细胞表位可以是诸如F21E的破伤风类毒素肽,包含肽序列FNNFTVSFWLRVPKVSASHLE(氨基酸947-967;SEQ ID NO:11)。
在另一个实施方式中,T辅助细胞表位被融合到至少一个抗原(即肽)或抗原的混合物来制备融合肽。
载体
所述组合物的载体包含疏水物质,优选液体疏水物质的连续相。所述连续相可以是基本纯的疏水物质或疏水物质的混合物。此外,载体可以是水在疏水物质中的乳液或水在疏水物质混合物中的乳液,前提是疏水物质构成连续相。并且,在另一个实施方式中,载体可以行使佐剂的功能。
此处所述的用于所述组合物的疏水物质是那些可药用的和/或免疫可接受的。载体优选为液体,但是在室温下不是液体但是可以被液化(例如通过加温)的疏水物质也用于本发明。在一个实施方式中,疏水载体可以是PBS/FIA乳液。
油或油包水乳液是尤其适宜用于本发明的载体。油应该是可药用的和/或免疫可接受的。油的优选例子为矿物油(尤其是轻的或低粘度矿物油)、蔬菜油(例如玉米或介花油)、坚果油(例如花生油)和角鲨烯。最优选为低粘度矿物油。动物脂肪和人工合成疏水聚合物材料,尤其是那些室温下为液体或相对容易被液化的也可以使用。
脂质体
脂质体是含有被包围的含水的容积的完全封闭的脂双层膜。脂质体可以 是单层囊泡(具有单个双层膜)或特征为多双层膜的多层囊泡,每个双层可以被含水层与相邻层隔开,也可以不被含水层与相邻层隔开。脂质体的综合讨论可见Gregoriadis G.Immunol.Today,11:89-97,1990;and Frezard,F.,Braz.J.Med.Bio.Res.,32:181-189,1999。
虽然任何脂质体,包括由原始细菌脂类制成的脂质体,都可以用于本发明中,但尤其有用的脂质体是在脂质体配方中使用磷脂类和未酯化的胆固醇。胆固醇用来稳定脂质体,稳定脂质体的任何其他化合物可取代胆固醇。其他脂质体稳定的化合物为本领域技术人员已知。例如,饱和的磷脂产生具有更高转变温度的脂质体,该高转变温度表明其有增加的稳定性。为了避免限制抗原和脂质体之间的静电联系,抗原可以被隔绝在脂质体的内部。
制备脂质体时优选使用的磷脂是那种具有选自由磷酸甘油、磷酸乙醇胺、磷酸丝氨酸、磷酸胆碱和磷酸肌醇所组成的组中的至少一个头部基团的磷脂。更优选的是含有卵磷脂Phospholipon90G中的脂类的脂质体。当在脂质体配方中还使用未酯化的胆固醇时,胆固醇的用量相当于磷脂量的大约10%。如果使用胆固醇之外的化合物稳定脂质体,本领域技术人员能够容易地确定在所述组合物中需要的量。
用天然脂类、合成脂质、神经鞘脂类、醚脂类、甾醇类、心磷脂、阳离子脂质和用聚乙二醇和其他聚合物修饰的脂类可以得到脂质体组合物。合成脂类可包括下列脂肪酸的构成部分:月桂酰、肉豆蔻酰、棕榈酰、硬脂酰、二十烷酰、油酰、亚油酰、伊鲁克酰(erucoyl)或这些脂肪酸的组合。
佐剂
所述组合物可进一步包含一种或多种可药用的佐剂、辅药等,如现有技术中已知的。例如,见《Remington’s Pharmaceutical Sciences》(Remington’sPharmaceutical Sciences,Mack出版公司,美国宾夕法尼亚州伊斯顿,1985年)和《美国药典》:1999年出版的美国药品集(USP 24 NF19)。在一个实施方式中,适宜的佐剂包括含CpG的寡聚脱氧核苷酸(CpG ODN)。例如 5′-TCCATGACGTTCCTGACGTT-3′。本领域技术人员可以基于目标种类和功效选择合适的CpG。代替CpG,也可使用脂肽,例如Pam3Cys-SKKK(EMCMicrocollections,德国)或其变体、同源体或类似物。在这一方面,脂肽的Pam2家族已经表现为脂肽的Pam3家族的有效替代物。
佐剂的使用量取决于抗原的量和佐剂的类型。本领域技术人员能够容易地确定在具体应用中所需的佐剂的量。
组合物
在一个实施方式中,此处所述的组合物可以通过如下方法来配制形成:在脂质体中胶囊化抗原(定义为与游离抗体和/或淋巴细胞上抗原结合受体特异性相互作用的物质)或抗原/佐剂复合物来形成脂质体胶囊化的抗原,并将该脂质体胶囊化的抗原与含有疏水物质连续相的载体混合。如果在第一步中不使用抗原/佐剂复合物,则在载体与脂质体胶囊化的抗原混合之前,可将适宜的佐剂加入脂质体胶囊化的抗原中,加入脂质体胶囊化的抗原和载体的混合物中,或加入载体中。该过程的顺序取决于所用佐剂的类型。然后得到的脂质体胶囊化的抗原与载体混合。(应注意,根据上下文,术语“脂质体-胶囊化的抗原”可指单独抗原的脂质体胶囊化或抗原/佐剂复合物的胶囊化。)这促进了佐剂和抗原的直接接触,并可以至少部分解释其良好的免疫反应。为了促进一些佐剂的应用,所述抗原可以先在脂质体中胶囊化,然后得到的脂质体胶囊化抗原与佐剂在含有疏水物质连续相的载体中混合。
在配制基本无水的组合物时,所述抗原或抗原/佐剂复合物可以用脂质体胶囊化,其可以是冻干的也可不是冻干的,并悬浮于疏水物质中。当在疏水物质包水的乳液中配制组合物时,所述抗原或抗原/佐剂复合物可以在脂质体中胶囊化,悬浮于含水介质中,然后将该含水介质与疏水物质混合形成乳液。在乳液的情况下,为了保持疏水物质处于连续相,所述含有脂质体的含水介质可以等分试样在混合下加入到该疏水物质中。
在一个实施方式中,所述抗原或脂质体胶囊化的抗原可在与疏水物质或 含水介质混合前冻干,根据情况而定。在另一个实施方式中,抗原/佐剂复合物可用脂质体胶囊化,然后再冻干。在进一步的实施方式中,抗原可以在脂质体中胶囊化,然后加入佐剂,再冻干,形成具有外部佐剂的冻干的脂质体胶囊化抗原。在又一种情况下,所述抗原可以用脂质体胶囊化,然后在加入佐剂前冻干。冻干可以促进佐剂和抗原之间的更好的相互作用。
在另一个实施方式中,脂质体胶囊化的抗原进入疏水物质的配方还可包括乳化剂的使用,以促进脂质体在该疏水物质中更均匀的分布。典型的乳化剂为本领域所公知,包括油酸二缩甘露醇酯(ArlacelTM A)、卵磷脂、TweenTM 80和SpansTM 20、80、83和85。乳化剂以能够有效促进脂质体更均匀分布的量使用。通常,疏水物质与乳化剂的体积比(v/v)范围为约5∶1~约15∶1,优选约10∶1。
另外,所述抗原或抗原/佐剂复合物可以不在脂质体中胶囊化,而与脂质体相结合,相接触,或与脂质体相分离。一些亲水性抗原或亲水性抗原/佐剂复合物的脂质体胶囊化的效率可能较差,所以在被放入疏水环境中或冻干时,大部分抗原与脂质体的外表面相结合。这代表了本发明的另一个实施方式。
在进一步的实施方式中,具有CTL表位和PADRE(融合到该抗原上或单独存在)的抗原(肽或多肽)可以一起被胶囊化到脂质体中。在另一个实施方式中,在同样的脂质体中可以一起放入多于一种的抗原。在进一步的实施方式中,可使用具有T辅助细胞表位的其他物质来代替PADRE,例如破伤风类毒素肽。在另一个实施方式中,佐剂,优选含CpG的ODN,也可在脂质体中胶囊化。优选该脂质体悬浮于PBS中。这种悬浮液随后在疏水载体,例如ISA51或矿物油中乳化。结果,含有抗原和佐剂,优选PADRE和CpG的脂质体被悬浮于PBS中,接下来在疏水载体,例如ISA51或矿物油中乳化。
癌症的复发总是人们会关心的问题,所以长效CTL反应的诱导对于保 证癌症不复发很重要。总的来说,CTL反应短期存在,仅仅维持几周,但是此处所述的组合物能够诱导持续至少30、40、50、60、70、80、90、100、110、120或130天的有效CTL反应。
在一个实施方式中,从事先用一种组合物处理后130天的小鼠分离的脾细胞保持了裂解小鼠淋巴瘤EL-4细胞的能力(图3),所述组合物含有在油包水乳液中悬浮的在脂质体中胶囊化的CpG ODN和融合到PADRE的HPV的E7蛋白的CTL表位。这些结果表明此处所述的组合物能够诱导长效CTL反应,这是癌症治疗所想要的。
在一个实施方式中,用含有CpG ODN佐剂和TRP-2和/或p53肽作为抗原的组合物的治疗能够增加产生在与癌细胞斗争中需要的抗原特异性干扰素γ(IFN-γ)的脾细胞的数量(图6-8)。抗原被融合到通用T辅助细胞表位(PADRE),并在悬浮于油包水乳液中的脂质体中胶囊化。肽和表达该肽来源蛋白的肿瘤细胞均能诱导IFN-γ的产生,说明输送肽抗原的本发明的组合物的使用导致了与预期靶点相关的免疫反应。
在另一个实施方式中,用此处所述的组合物的单独治疗对已建立的肿瘤进行的治疗在显著减小肿瘤大小和降低处理后带有肿瘤的小鼠的百分比方面是有效的(图9-13和15)。
用此处所述的组合物的治疗之后可以向给药位点用合适的组合物涂敷皮肤,所述组合物包含咪喹莫特,(1-(2-甲基丙基)-1H-咪唑并[4,5-c]喹啉-4-胺),或其类似物,其属于一类能够通过局部诱导细胞因子来激活免疫系统的非核苷咪唑并喹啉胺(杂环胺)。咪喹莫特是TLR7的配基,激活Th1样细胞因子环境,包括IFN-α、TNF-a、IL-1α、IL-6和IL-8。在进一步的实施方式中,用此处所述的组合物处理后,可以向给药位点的皮肤涂敷AldaraTM软膏(咪喹莫特5%)(3M,St.Paul,MN,U.S.A.)。
在一个实施方式中,在用组合物进行单次给药,然后在给药治疗的位点施以Aldara乳膏的皮肤涂敷来进行治疗的小鼠中,肿瘤大小和带瘤小鼠的 百分比降低了(图18和19),所述组合物含有在悬浮于油包水乳液中的脂质体中胶囊化的CpG ODN和融合肽。
此处所述的组合物可以被配制成适合口服给药、鼻腔给药、直肠给药或非消化道给药的形式。非消化道给药包括静脉给药、腹膜内给药、真皮内给药、皮下给药、肌肉注射、经上皮给药、肺内给药、鞘内给药和局部给药方式。优选途径为肌肉注射、皮下给药和真皮内给药,以达到储存效果。
当在单独涂敷给药时,此处所述的组合物也可有效。
在另一个实施方式中,此处所述的组合物可以在其他癌症治疗,例如放疗和化疗之前或之后联合应用。以前已经表明,当诊断为II期或III期黑色素瘤的病人外科手术治疗后给以含有诱导针对黑色素瘤特异性抗原的CTL反应的组合物的疫苗组合物时,能防止黑色素瘤的复发。(Antonia,S.J.et al.,Clin.Cancer Res.12:878-887,2006;Allegra,C.J.and R.W.Childs.,J.NationalCancer Inst.97:1396-1397,2005;Cassarino,D.S.et al.,J.Cutaneous Path.33:335-342,2006;Correale,P.et al.,J.National Cancer Inst.97:1437-1445,2005;Gulley,J.L.et al.,Clin.Cancer Res.11:3353-3362,2005;andChakraborty,M.et al.,Cancer Res.64:4328-4337,2004).
通过下面的非限制性实施例来进一步说明本发明。
实施例
实施例1
细胞反应
(a)激活
为检测CTL反应的特异性和速度,小鼠用组合物处理一次,该组合物含有CpG ODN佐剂、融合到通用T辅助细胞表位PADRE(AKXVAAWTLKAAA-OH(SEQ ID NO:10);50μg/剂)的人乳头状瘤病毒(HPV)16的CTL表位,即R9F(E7(H2-Db)肽RAHYNIVTF,氨基酸49-57;SEQID NO:1),上述物质在悬浮于PBS/FIA(磷酸盐缓冲液/弗氏不完全佐剂) 乳液(100μl/剂)中的脂质体(0.2g卵磷脂和0.02g胆固醇/剂)中胶囊化。处理后14天,脾细胞(效应细胞)与R9F或无关肽(KIMCNSSCM;SEQ ID NO:13)共培养6小时。脾细胞的离体胞内IFN-γ染色说明,脾细胞暴露于R9F肽时的IFN-γ阳性CD8+T细胞(CTLs)的比例(1.6%的脾细胞)是暴露于无关肽中时的13倍(0.12%的脾细胞或无肽;图1)。如图1所示,与用含有无关肽的组合物处理的小鼠相比,用上述含有R9F肽的组合物对小鼠的处理引起了对HPV表位刺激呈现特异性反应的CTLs的显著扩张。
胞内淋巴因子染色表明IFN-γ阳性CTL的存在。为了说明产生IFN-γ的CTL的保护功能,用含有CpG ODN和融合有PADRE的R9F肽并在悬浮于油包水乳液中的脂质体中胶囊化的组合物处理小鼠。处理后30天,携带有R9F肽(RAHYNIVTF;SEQ ID NO:1)的E4细胞(靶细胞;小鼠淋巴瘤细胞系)和携带有无关肽(KIMCNSSCM;SEQ ID NO:13)的E4细胞用来自处理过的小鼠的脾细胞体外刺激6天。用JAM检测测量细胞毒性(图2)。体外刺激6天后,大约50%的带有R9F肽的EL-4细胞(效应器与靶器官比率为10)被来自用含有R9F肽的组合物处理过的小鼠的脾细胞裂解(方块)。相反,仅有大约5%的带有无关肽的EL-4细胞被同样的脾细胞裂解(菱形;p<0.009)。
(b)持续期
用本发明的具体实施例单次处理诱导的记忆应答的持续期通过用下列组合物处理后130天的小鼠的脾细胞对EL-4细胞的裂解来说明(图3):(i)在PBS/FIA油包水乳液中,在脂质体中胶囊化的融合肽(R9F肽融合以PADRE)和CpG ODN(菱形);(ii)在PBS/FIA油包水乳液中,未胶囊化的融合肽和CpG ODN(三角形和叉形);或(iii)不含CpG ODN佐剂的在PBS/FIA油包水乳液中脂质体胶囊化的融合肽(实心圆圈)。用脂质体胶囊化的融合肽、脂质体胶囊化的CpG ODN、未胶囊化的融合肽和CpG ODN和未胶囊化的肽进行处理的脾细胞作为对照脾细胞。
JAM检测使用六天的体外刺激,然后将脾细胞(效应细胞)与预载了3H- 标记的胸腺嘧啶核苷的携带R9F或无关肽的EL-4细胞(靶细胞)共培养。当效应器对靶细胞的比例为25∶1和5∶1时,来自用在悬浮于油包水乳液中的脂质体中胶囊化的融合肽和CpG ODN免疫的小鼠的脾细胞裂解了30%的携带了R9F肽的靶细胞,而当该比例为1∶1时,裂解了15%的靶细胞(图3)。来自给以对照处理的小鼠的脾细胞表现出背景水平的细胞毒性。与文献中报道的CTL反应的持续期相比,在单次处理之后大于130天的CTL反应的持续期具有显著性。
实施例2
宫颈癌的根除
尽管已经开发了用于人乳头状瘤病毒(HPV)诱导的宫颈癌和外阴癌的预防性疫苗,例如GardasilTM和CervarixTM,但是宫颈癌和外阴癌的治疗性处理仍旧是高度优先的。在这个实施例中,含有人乳头状瘤病毒(HPV)16的CTL表位,即R9F(E7(H2-Db)肽RAHYNIVTF,氨基酸49-57;SEQ ID NO:1)的治疗组合物用于诱导CTL。这些CTL需要CD4+T细胞协助其分化和扩增,以及其成熟为功能化的记忆CTL。为了达到通过CD4+T细胞辅助的有效的CTL反应,R9F被融合到通用T辅助细胞表位,PADRE(SEQ ID NO:10),得到融合肽。该融合肽与含有CpG ODN基序或脂肽(Pam3Cys-SKKKK)的合成的寡聚脱氧核苷酸一起在脂质体中胶囊化。该治疗组合物利用PBS/FIA油包水乳液在单次处理中输送该治疗配方。为说明治疗性处理的效率,用表达HPV 16的C3肿瘤细胞来攻击C57BL/6小鼠(10只小鼠/组),然后在攻击后的第14天用上述治疗组合物或对照组合物来处理这些小鼠。到第30天(即处理后16天),在用C3肿瘤攻击然后给予处理的组中的所有10只小鼠都表现为明显肿瘤的完全消除(图4;空心方块)。相反,用除了融合肽以外含有上述治疗性处理中的所有成分的组合物处理的10只对照小鼠中的肿瘤尺寸继续增大(实心方块)。
实施例3
预防
为了进一步说明本发明的组合物保护对抗C3肿瘤细胞攻击的能力,对雌性C57BL/6小鼠在尾巴基部用含有用PADRE(SEQ ID NO:10)融合的R9F肽(RAHYNIVTF;SEQ ID NO:1)(称作融合肽)与CpG ODN在悬浮于油包水乳液中的脂质体中胶囊化的组合物进行皮下注射。为了确定该组合物是否与含有代替CpG ODN的取代佐剂的组合物一样有保护性,因此对小鼠给以如上所述的组合物,只是其中的CpG ODN可替代的CpG佐剂即Pam3c(Pam3Cys-sKKKK)代替。对照组用PBS、PBS中的CpG ODN、PBS中的融合肽、悬浮在含有CpG ODN的PBS中的融合肽或不含佐剂的在脂质体中胶囊化的融合肽。
单次处理后15天,0.5×106个C3细胞被皮下移植到处理过的小鼠的左侧腹,作为初次攻击(图5)。所有用PBS或溶于PBS的CpG ODN注射的小鼠在两周内都生长了肿瘤,并且根据动物饲养方案的需要,在第30天之前这些动物不得不因为肿瘤大小而退出该研究。溶于PBS中的融合肽的处理仅保护了20%的小鼠(处理组3)。在PBS乳液中的脂质体中胶囊化的融合肽使50%的小鼠没有生长肿瘤(处理组4),暗示脂质体胶囊化的融合肽提供了对于C3攻击的保护。用含于PBS乳液中的融合肽和CpG ODN的处理防止了60%的小鼠生长肿瘤(处理组5)。比较起来,用在悬浮于油包水乳剂中的脂质体中胶囊化的融合肽和CpG ODN处理的100%的小鼠在受攻击后61天的监测期内仍没有发生肿瘤(处理组6)。为了确定持续期和直接针对肿瘤的记忆应答的强度,对第6处理组中的小鼠给以6×106个C3肿瘤细胞的二次攻击。又过了73天,所有的小鼠还是没有肿瘤,说明用本发明的组合物的单次处理提供了稳健的长效的细胞免疫反应。同样,用Pam3c取代CpG,在所有的小鼠中也是61天没有发生肿瘤(处理组7)。这些小鼠没有用C3细胞再次攻击。
治疗
为了评价已建立的明显的C3肿瘤的治疗,对小鼠在左侧腹用0.5×106 个C3细胞进行皮下移植。在肿瘤移植后的第4、5、6或9天,对小鼠(n=10)用组合物和空白对照剂(PBS乳状液中的融合肽和CpG ODN)进行处理,所述组合物含有CpG ODN和用PADRE(SEQ ID NO:10)融合的R9F肽(RAHYNIVTF;SEQ ID NO:1)(融合肽),并在脂质体中胶囊化,悬浮于PBS/FIA油包水乳液中。单次免疫在第40天前消除了在肿瘤移植后第4、5或6天进行免疫的处理组中的所有10只小鼠和在肿瘤移植后第9天进行免疫的组中所有30只小鼠中的肿瘤。在肿瘤移植后第5天处理的组中仅有一只小鼠直到第40天还有肿瘤(表2)。相反,在肿瘤移植后第4或6天用安慰剂组合物处理的组中10只小鼠中有9只小鼠生长了肿瘤。在攻击后第5天用空白对照剂给药的小鼠的组中,10只小鼠全部生长了肿瘤,在肿瘤移植后第9天用空白对照疫苗处理的组中,30只小鼠中的27只生长了肿瘤。
为了评价用Pam3c代替CpG ODN是否会改变组合物消除C3肿瘤的能力,对10只小鼠用含有融合肽和Pam3c,在脂质体中胶囊化并悬浮于PBS/FIA油包水乳液中的组合物进行处理,或用空白对照剂处理(除不含脂质体外包含同样的成分)第二组的10只小鼠。然后对两个处理组中的小鼠在其左侧腹用1×106个C3细胞进行攻击。在脂质体中胶囊化的融合肽和Pam3c对已建立的C3肿瘤的治疗性处理重复两次,得到如表2所示的相似结果。
表2.在用以CpG ODN或Pam3c(*)作为佐剂的含有用PADRE(SEQ ID NO:10)融合的R9F肽(SEQ ID NO:1)(融合肽),在脂质体中胶囊化并悬浮于油包水乳液中的组合物或用含有除了脂质体外的所有上述成分的空白对照剂 疫苗处理的小鼠中的肿瘤的消除。
实施例4
黑色素瘤的治疗处理
酪氨酸酶是在黑色素瘤中过表达的一种蛋白。来自酪氨酸酶蛋白的肽一般是黑色素瘤治疗中比较差的抗原。如此处所述,V8L,结合到鼠科动物MHC,H2K2和人HLA-A2.1的一种来自酪氨酸酶相关蛋白(TRP-2)(氨基酸181-188;VYDFFVWL;SEQ ID NO:6)的肽被用于治疗性处理中来刺激产IFN-γ细胞的产生。TRP-2特异性的产IFN-γ细胞数量的刺激说明可以预期专门针对黑色素瘤的治疗性效果。
用本发明的含有在脂质体中胶囊化并悬浮于油包水乳液中的CpG ODN和融合了PADRE的TRP-2肽的组合物处理C57BL小鼠一次。对照处理采用不含CpG ODN的包含脂质体胶囊化的带有PADRE的TRP-2肽的组合物,和含有脂质体胶囊化的CpG ODN和带有PADRE的无关肽(KIMCNSSCM;SEQ ID NO:13)的组合物。在这两个对照处理中,脂质体悬浮在PBS/ISA51油包水乳液中。用ELISPOT对产IFN-γ脾细胞的离体检测表明,该处理组合物产生了最大数量的TRP-2特异性产IFN-γ细胞(图6,A组)。对照处理(图6,B组)产生了大约一半数量的TRP-2特异性产IFN-γ细胞,而用无关肽代替TPR-2的组产生了背景水平的TRP-2特异性产IFN-γ细胞(图6;group C),这些表明本发明的处理组合物,产生了最大数量的在对抗黑色素瘤中需要的TRP-2特异性产IFN-γ细胞。
实施例5
乳腺癌的治疗处理
p53基因产物对于治疗恶性肿瘤,尤其是乳腺癌是一个理想的并且广泛表达的靶点。大部分的人类癌症表现出p53突变,其为肿瘤发生的一个早期事件。p53的过表达是多数侵略性癌症、淋巴结转移肿瘤、基本治疗方案失败和最终癌症相关的死亡的独立预报因子。
用本发明的组合物单独处理的小鼠(图7;组A)产生了为用对照组合物处理的小鼠大约10~40倍的p53肽特异性产IFN-γ细胞,其中本发明的组合物中含有在的带有修饰的p53 CTL表位,mK9M,(KYICNSSCM;SEQ IDNO:8)与CpG ODN和PADRE(SEQ ID NO:10)的在PBS/ISA51油包水乳液中的脂质体中胶囊化,对照组合物分别是含有融合到PADRE的肽和CpGODN,但不含脂质体的组合物(图7;组B);含有在脂质体中胶囊化的融合肽但不含CpG ODN的组合物;或用无关肽代替融合肽的组合物(图7;组D)。肿瘤特异性产IFN-γ细胞的增加的产量与宫颈癌肿瘤的减小/消除相关(见实施例2和3),所以本领域技术人员应能够预测带有p53的肿瘤的相似结果。
实施例6
针对多于一个靶点的治疗性癌症处理
一些癌症同时表达多于一种的肿瘤相关蛋白。这种癌症提供多于一种的治疗性处理的靶点。例如,黑色素瘤细胞过表达p53和TRP,这提高了同时针对p53和TRP的治疗更加有效和特异的可能性,因为表达p53和TRP靶点的细胞对于处理会更加易感。
用组合物单独处理的小鼠产生了大约相等数量的p53和TRP特异性的产IFN-γ细胞(图8;组A),所述组合物含有带有CpG ODN和PADRE(AKXVAAWTLKAAA,其中X=环己基丙氨酰);SEQ ID NO:10)的p53(mK9M;KYICNSSCM;SEQ ID NO:8)和TRP-2(V8L;VYDFFVWL;SEQ IDNO:6)肽的混合物,并在悬浮于油包水乳液中的脂质体中胶囊化。相反,用没有脂质体的含有融合到PADRE的p53和TRP-2CTL肽的带有PADRE和CpG ODN的混合物处理的对照小鼠产生了比p53特异性产IFN-γ细胞更多的TRP-2-特异性产IFN-γ细胞(图8;组C),甚至在没有CpG ODN的情况下也是如此(图8;组B)。p53-特异性产IFN-γ细胞的产量为用对照处理得到的水平(即没有CpG ODN(组B)或没有CpG ODN和脂质体(组D)的处理组合物)。这些结果表明用本发明的处理组合物给药的小鼠具有对带有TRP-2 和p53肿瘤相关蛋白的肿瘤的双向攻击。尽管用TRP-2和p53两种肽进行了处理,但是没有在悬浮于油包水乳液中的脂质体中胶囊化的带有CpG ODN的融合肽处理的小鼠仅仅攻击一个靶点,TRP-2肿瘤相关蛋白。
实施例7
使用HLA A2转基因小鼠,其具有人HLA A2主要组织相容性复合体(MHC)基因,所以表达人MHC,所以其更好地模拟了人宫颈癌。为了与HLAA2 MHC相容,使用了不同于前面的实施例中使用的CTL表位。HLA A2小鼠用下列组合物之一进行处理:
(1)四个E6/7人乳头状瘤病毒(HPV)来源的肽(MP)的混合物,各个肽的序列如下:
Y10T(E7:氨基酸11-20;YMLDLQPETT;SEQ ID NO:2);
L9V(E7:氨基酸82-90;LLMGTLGIV;SEQ ID NO:3);
T81(E7:氨基酸86-93;TLGIVCPI;SEQ ID NO:4);和
T10V(E6:氨基酸29-38;TIHDIILECV;SEQ ID NO:5);
2)上述四个肽用“aay”连接体连接成一个长肽(AB2;SEQ ID NO:14),其序列如下:
TIHDIILECVaayYMLDLQPETTaayLLMGTLGIVaayTLGIVCPI;
或
3)选自上面所列四个肽的单一肽,即L9V(E7:氨基酸82-90;LLMGTLGIV;SEQ ID NO:3)。
所有的处理组合物包含PADRE(25μg/剂)和CpG ODN(50μg/剂)作为佐剂,并在PBS/ISA51油包水乳液中悬浮的脂质体中输送。每次处理中四个肽的混合物中每种肽含有25μg。所述长肽(AB2)以每次处理100μg给药。仅含有L9V的组合物以每次处理含有25μg给药。对照小鼠用PBS注射(每次处理100μL)。
在左侧腹皮下移植TC1/A2肿瘤细胞(1×105个细胞/小鼠)来攻击小鼠, 每5天测量肿瘤大小。攻击19天后,用上述组合物中的一种处理小鼠(5只小鼠/组),或用PBS注射(对照)。
如图9所示,含有上述四种HPV E6/7肽的混合物(方块)或AB2长肽(菱形)的组合物的单次处理在处理后21天消除了TC1/A2肿瘤。用含有E7肽L9V的组合物的处理显著减小了肿瘤大小(三角形)。只用PBS处理(叉形)不能防止肿瘤的生长。在对照组中注射PBS的五只小鼠中的肿瘤生长相似(图10)。因为其肿瘤尺寸过大,根据要求这五只小鼠在第35天退出研究。所报告肿瘤尺寸为五只小鼠的平均肿瘤尺寸。
用含有肽混合物的组合物处理的小鼠的肿瘤尺寸的减小是变化的(图11)。例如,小鼠2中的肿瘤在处理后第6天消除。剩下的小鼠中的肿瘤直到处理后至少第11天才消除。用含有长肽(AB2)的组合物处理的小鼠的肿瘤尺寸的减小也是变化的(图12)。然而,到处理后21天,所有五只小鼠中的肿瘤都完全消除。
在用含有单一HPV E7肽(L9V;SEQ ID NO:3)的组合物处理的小鼠中,肿瘤尺寸的减小,在五只小鼠中的四只中是相似的(图13)。小鼠3的处理并未导致肿瘤大小的减小,这暗示着用含有多于一个HPV肽,要么是单个肽的混合物,要么是融合肽,这样的组合物进行处理与针对单个肽的免疫比起来,会保护群体中更多的个体。
实施例8
在实施例7中,四个HPV 16 E6/E7肽用连接体“aay”(-丙氨酸-丙氨酸-酪氨酸-)连接成一个长肽。该连接体本质是疏水的,增加了融合长肽的疏水性,使得该肽难以制造并且需要使用二甲基亚砜来使该长肽可溶。
在本实施例中,连接体“aay”用连接体“kkp”(-赖氨酸-赖氨酸-脯氨酸-)代替形成2个二肽。一个二肽是Y10T-kkp-L9V(TIHDIILECVkkpLLMGTLGIV;SEQ ID NO:15),另一个是T81-kkp-T10V(TLGIVCPIkkpYMLDLQPETT;SEQ ID NO:16)。“kkp”连接体的使用得到了使疫苗的制造 更容易的亲水性融合肽。使用“kkp”连接肽产生的产IFNγ脾细胞与当同样的四个肽独立地(即,未连接)使用时得到的数量基本相同(图14)。这些结果说明使用kkp连接体促进了疫苗抗原的制造而不改变抗原加工和肽特异性的产IFNγ脾细胞的诱导。这些细胞的产生是有效消除癌细胞的良好指示剂。
实施例9
对黑色素瘤的治疗性处理
实施例4、5和6说明了本发明的组合物增加TRP-2和p53特异性产IFN-γ脾细胞的产量,进而建立针对黑色素瘤-相关蛋白的细胞免疫反应的刺激。B16-F10细胞(10×103个细胞/小鼠)被皮下移植到无病原体的C57BL/6小鼠的左侧腹。移植时小鼠为6~8周龄,在滤盖条件下,水和食物无限制供应的条件下饲养。黑色素瘤细胞移植后5天,小鼠接受用含有两种源自TRP-2的肽(V8L或S9L(SEQ ID NO:6 or 7);25μg/小鼠)之一,一种修饰的源自p53的肽(mK9M(SEQ ID NO:8);25μg/小鼠),或这些肽的混合物的组合物(每种肽25μg/小鼠)通过皮下注射进行单独处理。所有的组合物还包含PADRE(25μg/小鼠)和CpG ODN(50μg/小鼠),并在悬浮于PBS/ISA51油包水乳液中的脂质体中输送。对照小鼠仅仅接受PBS给药。所有的注射都在尾巴根部给药。每4~5天用下列公式测定肿瘤大小:最大测量值×(最小测量值)(Pilon-Thomas et al.,J.of Immunother.,29(4),2006)。
图15说明用或者含有V8L(SEQ ID NO:6)(菱形)或者含有mK9M肽(SEQ ID NO:8)(三角形)的组合物处理的小鼠最初抑制黑色素瘤细胞的生长,但是黑色素瘤细胞克服了这种最初的生长抑制,形成了长到1200mm3的肿瘤。用含有S9L肽(SEQ ID NO:7)(方块),或V8L和mK9M(KYICNSSCM)的混合物(叉形),或S9L和mK9M的混合物(星形)的组合物的处理在整个监测期间抑制了黑色素瘤生长。单纯PBS(圆圈)对肿瘤生长没有影响。
在本研究结束时,带瘤小鼠的百分比表明含有mK9M(SEQ ID NO:8)(三 角形)、V8L(SEQ ID NO:6)(菱形)、或S9L(SEQ ID NO:7)(方块)的本发明的疫苗仅分别治愈了0、40和40%的小鼠的肿瘤(图16)。相反,用含有S9L和mK9M的混合物的组合物的处理(星形)治愈了80%的小鼠的肿瘤,含有V8L和mK9M的混合物的组合物(叉形)治愈了100%的小鼠的肿瘤。
实施例10
B16黑色素瘤肿瘤模型
在前面的实施例中,在两个独立的HPV-宫颈癌模型(C3和TC1/A2)的已建立的肿瘤中说明了本发明的组合物的有效性。通过靶向呈现在肿瘤细胞表面的HPV的CTL表位消除了带有HPV的肿瘤。在治疗病毒诱导的癌症时这一策略特别有效。但是呈现过表达的“自体”抗原的肿瘤难以治疗,因为它们不能被免疫系统发现。自体抗原被耐受机制紧密保护。有效治疗癌症的治疗必需具有诱导针对过表达的肿瘤相关的自体抗原的免疫反应的能力。相信黑色素瘤(包括B16肿瘤模型)下调了I类MHC的表达和自体抗原的呈现。用于治疗黑色素瘤的治疗性组合物必需激活能够靶向肿瘤表面的自体表位的低亲和性T细胞克隆型。
通过接种疫苗对黑色素瘤的成功治疗需要稳健的特异性的CTL反应。在临床前研究中,已表明B16特异性CTL活性在活体内不足以防止B16肿瘤的生长(Bellone et al.,J.of Immunol.,165(5):2651-2656,2000)。用来自TRP-2的黑色素瘤相关的自体表位脉冲冲击的CpG-成熟的树突细胞的免疫治疗不能使肿瘤的消退(Pilon-Thomas et al.,J.of Immunother.,29(4),2006)。在两个其他研究中,5日龄的已建立的B16肿瘤的治疗使少于50%的治疗的小鼠的肿瘤消除,而所有治疗的动物中的肿瘤都再次出现(Pilon-Thomas etal.,J.of Immunother.,29(4),2006;and Bronte et al.,Cancer Res.,60:253-258,2000)。
检测了本发明的组合物提高同时针对多个肽抗原的有效CTL反应的能力。小鼠(5只/组)用104个B16细胞移植,移植后6天用每次处理含有(0.1ml/ 剂)25μg的TRP-2CTL表位(S9L;SVYDFFVWL SEQ ID NO:7),25μg的p53CTL表位(mK9M;KYICNSSCM SEQ ID NO:8),25μg的PADRE和50μg的CpG ODN的组合物处理一次。作为对比,第二组小鼠(5只/组)用每次处理含有25μg同样的TRP-2CTL表位或25μg的p53CTL表位(K9M;KYMCNSSCM SEQ ID NO:9)、25μg修饰的的p53CTL表位、mK9M(SEQID NO:8)、25μg的PADRE(AKXVAAWTLKAAA,其中X=环己基丙氨酰;SEQ ID NO:10)和50μg的CpG ODN的组合物处理。所述组合物的所有成分在疏水载体ISA51中乳化之前加入脂质体中。对照小鼠只用PBS处理。
用含有TRP-2和p53表位混合物的组合物进行单独处理,在处理后21天,消除了所有的小鼠的肿瘤(图17;三角形),而用只含有TRP-2(菱形)或只含有p53(方块)的组合物的处理仅仅在40%的小鼠中清除了肿瘤。所有用PBS注射的对照小鼠都生长了肿瘤(叉形)。
实施例11
用C3肿瘤攻击小鼠(C57BL/6),在攻击后8天肿瘤生长为可触知的大小。在攻击后第8天,小鼠被分为两个对照组(10只/组),并且处理组用含有在油包水乳液中悬浮的脂质体中胶囊化的CpG ODN和融合肽(R9F肽(SEQID NO:1)融合到PADRE(SEQ ID NO:10))的组合物进行单次处理。
处理后15~20小时内,对小鼠在处理给药的位点用AldaraTM软膏(25mg(相当于10~12μl的Aldara))进行皮肤涂敷。在Aldara中的活性成分是浓度为5%的咪喹莫特。咪喹莫特(1-(2-甲基丙基)-1H-咪唑并[4,5-c]喹啉-4-胺)是已知具有局部免疫反应改性剂和刺激剂性质的咪唑并喹诺酮药物家族中的一种新型的合成化合物。咪喹莫特是TLR7的配基,并激活包括IFN-α、TNF-α、IL-1α、IL-6和IL-8的Th1样细胞因子环境。相反,两个对照组仅仅接受PBS处理(100μL/小鼠)或用PBS处理后再用Aldara软膏(25mg)进行皮肤涂敷。用治疗组合物处理后再用Aldara进行皮肤涂敷的小鼠中的肿瘤尺寸减小(图18;方块),而仅用PBS处理(叉形)的组中的小鼠和用PBS 处理后再用Aldara涂敷(三角形)的小鼠中的肿瘤尺寸没有减小。图19表明在用治疗组合物处理后再用Aldara进行皮肤涂敷(菱形)的组中带瘤小鼠的百分比减小,但是仅给以PBS(三角形)或给以PBS之后用Aldara涂敷(方块)的组中该百分比都没有减小。
实施例12
用破伤风类毒素肽F21E作为T辅助细胞表位的黑色素瘤治疗
黑色素瘤相关抗原,TRP-2,与源自破伤风类毒素的T辅助细胞表位一起在含有CpG ODN的组合物中在PBS/ISA51油包水乳液中被一起胶囊化。破伤风类毒素肽代替在前面的实施例中使用的T辅助细胞表位PADRE,来说明科使用的各种T辅助细胞表位。对TRP-2肽特异性产IFN-γ细胞数量的刺激表明特异性针对黑色素瘤的治疗效果是可预期的。
用含有TRP-2肽(S9L;SEQ ID NO:7)和破伤风类毒素表位F21E(氨基酸947-967,FNNFTVSFWLRVPKVSASHLE;SEQ ID NO:11)在带有CpGODN的脂质体中一起胶囊化的的组合物免疫C57BL小鼠。用上述组合物配方但其中不含破伤风类毒素T辅助细胞表位,免疫对照小鼠。在分离自免疫后8天采集的脾的脾细胞上,用ELISPOT进行IFN-γ的离体检测。对照和处理过的小鼠的脾细胞以每孔5×105个细胞铺细胞板,并用TRP-2肽(S9L)或用黑色素瘤细胞系B16-F10(5×104个细胞每孔,效应器对靶点的比例为1∶10)进行体外刺激。用治疗组合物免疫的小鼠的脾细胞含有最大数量的TRP-2特异性产IFN-γ细胞。当用TRP-2肽或B16-F10细胞刺激脾细胞时,观察所述免疫反应(图20)。对照组小鼠的脾显示出背景水平的TRP-2特异性产IFN-γ细胞。所以,当脾细胞在破伤风类毒素表位存在下用TRP-2肽刺激时,以及用B16-F10细胞表面呈现的黑色素瘤抗原刺激时,可检测到强的抗黑色素瘤CTL免疫反应。
方法
细胞系
C3细胞系在添加有10%热灭活的胎牛血清(Sigma,St Louis,MO),2mML-谷氨酰胺(Gibco,Burlington,ON),50mM 2-巯基乙醇(Gibco,Burlington,ON),100U/ml青霉素和100μg/ml链霉素(Gibco,Burlington,ON)的Iscove改良的达尔贝科氏培养基(IMDM;Sigma,St Louis,MO)中培养。细胞在37℃/5%CO2下孵育。
EL-4细胞系是来源于小鼠的淋巴瘤细胞系。EL-4细胞系在含有2mM L-谷氨酰胺,添加了10%热灭活的胎牛血清(Sigma,St Louis,MO),50mM 2-巯基乙醇(Gibco,Burlington,ON),100U/ml青霉素和100μg/ml链霉素(Gibco,Burlington,ON)的高糖含量的达尔贝科氏改良的伊格尔培养基(DMEM;Sigma,St Louis,MO)中保持培养。细胞在37℃/5%CO2下孵育培养。
B16F1(B16)黑色素瘤细胞系购自维吉尼亚州马纳萨斯的美国典型培养物保藏中心(ATCC)。
肽
由Dalton Chemical Laboratories有限公司(Toronto,ON)将含有CTL表位的HPV 16 E7(H-2Db)肽RAHYNIVTF49-57(R9F)与含有CD4+辅助细胞表位(CD4+helper epitope)的PADRE融合。该融合肽以50μg/剂使用。其中指明,R9F用作抗原(25μg/剂)或用于细胞毒性检测。该肽KYMCNSSCM(SEQID NO:13)(Dalton)被用作无关对照肽。
酪氨酸酶相关蛋白(TRP-2)肽S9L(氨基酸180-188;SVYDFFVWL;SEQID NO:7)和V8L(氨基酸181-188;VYDFFVWL;SEQ ID NO:6),以及p53肽(野生型p53(K9M),氨基酸232-240;KYMCNSSCM;SEQ ID NO:9),修饰的p53肽mK9M(氨基酸232-240;KYICNSSCM;SEQ ID NO:8)和连接到PADRE(AKXVAAWTLKAAAKYICNSSCM;SEQ ID NO:17)的mK9M购自Dalton Chemical Laboratories有限公司(Toronto,ON,Canada)。这些肽由鼠的I类MHC H-2K提供。S9L也由MHC HLA A2提供,然而,V8L不由MHCHLA A2提供。TRP2和p53肽在DMSO中以1mg/ml的储存液储存。用PBS 进行用于疫苗制造的进一步稀释。
除了那些含有结合的mK9M的所有的疫苗配方都含有PADRE(25μg/剂)和CpG ODN 1826(50μg/剂)。因为结合的mK9M含有PADRE作为其结构的一部分,所以,不需再加入游离的PADRE。
在ELISPOT测定中使用的无关肽的氨基酸序列是KIMCNSSCM(DaltonChemical Laboratories有限公司)。
佐剂
CpG ODN(带有下划线的CpG基序的合成的ODN 18265′-TCCATGACGTTCCTGACGTT-3′,50μg/剂)(SEQ ID NO;12)购自ColeyPharmaceutical(Wellesley,MA)。脂肽(Pam3Cys-SKKKK,(100μg/剂)购自德国EMC Microcollections。
处理
脂质体按如下所述制备:比例为10∶1的卵磷脂和胆固醇(0.2g卵磷脂和0.02g胆固醇/剂)溶于氯仿/甲醇(1∶1;v/v),并用PTFE 0.2μm过滤器无菌过滤该溶液。用旋转蒸发器减压去除氯仿和甲醇,在真空中进一步从得到的薄脂层中去除痕量溶剂。为了脂质体的胶囊化,带有CpG的融合肽溶于无菌PBS中,得到的溶液在搅拌下加入到薄脂层中形成脂质体。通过将该脂质体/PBS悬浮液加入到FIA中形成油包水乳液(PBS∶FIA;1∶1,v/v;100μl/剂),使得到的脂质体的悬浮液在FIA(Sigma,St Louis,MO)中被乳化。在一些实验中,使用Montanide ISA51(Seppic,France)取代FIA作为油载体。
购自Charles River Laboratories(Wilmington,MA)的无病原体的C57BL/6雌性小鼠,6~8周龄,在有滤盖的条件下,给以无限制的水和食物进行饲养。所有的实验都遵守动物饲养和使用规章。通过在尾巴根部皮下注射来用本发明的组合物处理小鼠。除非另外说明,所有的处理都是单次处理,并且所有的处理组均包括10只小鼠。对照小鼠皮下注射PBS或融合肽(融合到PADRE的选择的CTL表位)、R9F肽、CpG ODN(或Pam3c)、PBS中的 带有CpG的融合肽(100μl)、或在油包水溶剂(PBS/FIA;1∶1,v/v,100μl/剂)中胶囊化融合肽、R9F、CpG(或Pam3c)的脂质体。
肿瘤移植
在肿瘤移植中使用的C3细胞生长至95%汇合,用0.05%胰蛋白酶消化收获细胞。为了在小鼠中建立肿瘤,用0.5×106个C3细胞在小鼠左侧腹注射。每4~5天用下列公式测定肿瘤大小:最大测量值×最小测量值2再除以2。
通过在左侧腹皮下移植TC1/A2肿瘤细胞(1×105个细胞/小鼠)来攻击小鼠(HLA A2)。每5天测量肿瘤大小,报告单个小鼠的肿瘤大小和带瘤小鼠的百分率。
B16-F10细胞(10×103个细胞/小鼠)被皮下移植到没有病原体的C57BL/6小鼠的左侧腹,在移植时小鼠为6~8周龄。每2~5天测量肿瘤大小,结果报告为带瘤小鼠百分率。
细胞毒性检测
CTL检测、ELISPOT和干扰素(IFN)-γ的胞内染色表明治疗性反应对于选择的E7肽是特异性的,因为无关肽不会引起高于背景的CTL活性或IFN-γ产量。这些研究表明在来自给以治疗性处理的小鼠的脾细胞中,激活的特异性处理的细胞毒性T细胞的增加与肿瘤尺寸的减小相关。所用方法的详细过程如下所述。
淋巴母细胞产生和体外刺激(IVS)
为了检测急性和记忆性CTL反应,除非另外说明,分别在免疫后7、14或130天分析来自处理过的小鼠的脾细胞。如上所述,在一轮IVS之后进行细胞毒性检测。简单地说,体外刺激前3天,通过CO2窒息处死初次进行实验的C57BL/6小鼠,收获并分离脾。在RPMI-10中洗涤并计数脾细胞,其中RPMI中添加以10%热灭活的胎牛血清(Sigma,St Louis,MO),50mM 2-巯基乙醇(Gibco),100U/ml青霉素和100μg/ml链霉素(Gibco)。脾细胞(106个 细胞/毫升)与脂多糖(25μg/ml)和硫酸葡聚糖(7μg/ml)处理的淋巴母细胞一起培养。
辐照同源的淋巴母细胞(137Sc源辐照15分钟,剂量为4000rad)并载以R9F肽(100μM)。载肽的LPS激活的淋巴母细胞(3×106个细胞/毫升)用于以3∶1的比例刺激免疫的小鼠的脾细胞,其中效应器细胞被调节为3×106个细胞/毫升,向孔中加入T-stim(BD Biosciences,Mississauga,ON),使其终浓度为20%。在37℃/5%CO2下培养细胞6天。
JAM检测
用5μCi/ml[甲基-3H]胸腺嘧啶脱氧核苷(Amersham Pharmacia,Erlangen,Germany)标记EL-4细胞。在37℃/5%CO2孵育细胞24小时,然后载以R9F或无关肽(10μg/ml)一小时。然后收获标记的靶细胞悬液,在RPMI-10中洗涤两次,以2×103个细胞/孔接种于96孔U形底平板中。通过梯度稀释加入起始浓度为2×105个效应器细胞/孔的效应器细胞。该平板在37℃/5%CO2孵育4小时。将细胞吸到玻璃纤维滤膜上,并用Packard TopCount闪烁计数器对氚进行计数。DNA片段百分数用下列公式计算:%DNA片段=(S-E)/E×100,其中S为未经处理的(天然的)条件下保留的DNA(计数),E为效应器细胞存在的条件下保留的DNA(计数)。
用ELISPOT进行的抗原特异性的T细胞离体分析
用BD ELISPOT(BD Bioscience,San Diego,CA)检测处理过的C57BL/6小鼠中收获的脾细胞中的活化的抗原特异性CTL。简要地说,处理后第7天,用捕获抗体,纯化的抗鼠IFN-γ抗体包被96孔硝酸纤维素板,并在4℃孵育过夜。弃去抗体,封闭该板2小时,然后去掉封闭溶液。以100万个细胞/孔的初始浓度向各个孔中加入脾细胞,终体积为100μl,然后在一行中后面的孔中进行梯度稀释。接下来向该100μl的培养基中加入刺激剂和对照,以得到其所需的终浓度。C3细胞(5×105个细胞/毫升)、R9F肽(10μg/ml)或无关肽(10μg/ml)被加入到这些孔中,或不加入肽。PMA(5ng/ml,Sigma)、离 子霉素(500ng/ml,Sigma)作为阳性对照,无关肽和单独的培养基作为阴性对照。该平板在37℃/5%CO2孵育过夜,然后检测抗体,生物素标记的抗鼠IFN-γ抗体,室温下加入2小时。孵育期之后,弃去该检测抗体,加入酶交联物(抗生蛋白链菌素-HRP)1小时,最后该平板用AEC底物溶液染色20分钟。洗涤该平板,空气干燥过夜,用放大镜观察斑点。
胞内细胞因子染色(ICS)
如前所述,脾细胞从没有肿瘤的小鼠中取得,用RPMI-10洗涤两次(500×g,5分钟),在RMPI-10(10×106个细胞/毫升)中重悬。脾细胞(1×106 个细胞/孔)加入到96孔平底培养板中,与终浓度为3μg/ml的R9F或无关肽共孵育,每种肽两列。在用EL-4细胞说明产IFNγ的CD8+CTL的保护功能的实验中,加入R9F或无关肽的EL-4细胞(1×105个细胞/孔)在细胞毒性检测之前在37℃/5%CO2条件下孵育6小时。
按照CytofixTM/CytopermTM试剂盒说明书(BD Biosciences,Mississauga,ON)进行胞内细胞因子染色。简单地说,加入刺激物后,向每个孔中加入GolgiStop,该平板孵育(37℃/5%CO2)4小时。用染色缓冲液洗涤细胞,然后与抗-CD8血清一起孵育(4℃,暗处20分钟),再次用染色缓冲液洗涤,然后与抗-IFN-γ一起孵育(4℃,暗处30分钟)。然后用perm/洗涤缓冲液洗涤,然后用perm/wash缓冲液重悬,并转移到FACS试管中(BD Falcon)。用FACSCalibur(BD Biosciences,San Jose,CA)检测染色结果,用CellQuest软件分析数据。
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本申请文件中引用的所有出版物和专利申请在此通过引用而合并,就像每个单独的出版物或专利申请被特别地并且个别的说明为通过引用而合并一样。任何出版物的引用是因为其公开是在申请日之前,不应解释为承认本发明不能因为先发明而早于这些出版物。
虽然为了清楚理解的目的已经通过图示和实施例描述了上述发明,对本领域技术人员来说明显的是在本发明的教导下,可以对其进行某种改变和修饰而不背离所附权利要求书的范围或精神。
应注意的是,在本申请文件和所附权利要求书中,除非上下文清楚表明,否则单数形式″a,″″an,″和″the″包括所指代物的复数。除非另外限定,此处使用的所有技术和科学术语具有本发明所属技术领域的普通技术人员一般理解的相同意义。
序列表
<110>免疫疫苗技术有限公司
<120>脂质体在含连续疏水相的载体中作为癌症治疗媒介物的应用
<130>78961-49
<150>CA 2,523,032
<151>2005-10-07
<150>CA 2,533,705
<151>2006-01-13
<150>CA 2,542,212
<151>2006-04-07
<160>55
<170>PatentIn version 3.3
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<213>未知
<220>
<223>黑色素瘤相关抗原
<400>7
Ser Val Tyr Asp Phe Phe Val Trp Leu
1 5
<210>8
<211>9
<212>PRT
<213>未知
<220>
<223>黑色素瘤相关抗原
<400>8
Lys Tyr Ile Cys Asn Ser Ser Cys Met
1 5
<210>9
<211>9
<212>PRT
<213>未知
<220>
<223>黑色素瘤相关抗原
<400>9
Lys Tyr Met Cys Asn Ser Ser Cys Met
1 5
<210>10
<211>13
<212>PRT
<213>未知
<220>
<223>通用T辅助细胞表位(PADRE)
<220>
<221>misc_feature
<222>(3)..(3)
<223>Xaa=环己基丙氨酰
<400>10
Ala Lys Xaa Val Ala Ala Thr Thr Leu Lys Ala Ala Ala
1 5 10
<210>11
<211>21
<212>PRT
<213>未知
<220>
<223>T辅助细胞表位(F21E)
<400>11
Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser
1 5 10 15
Ala Ser His Leu Glu
20
<210>12
<211>20
<212>DNA
<213>未知
<220>
<223>佐剂
<400>12
tccatcacgt tcctgacgtt
20
<210>13
<211>9
<212>PRT
<213>未知
<220>
<223>无关肽
<400>13
Lys Ile Met Cys Asn Ser Ser Cys Met
1 5
<210>14
<211>46
<212>PRT
<213>未知
<220>
<223>连接的肽
<400>14
Thr Ile His Asp Ile Ile Leu Glu Cys Val Ala Ala Tyr Tyr Met Leu
1 5 10 15
Asp Leu Gln Pro Glu Thr Thr Ala Ala Tyr Leu Leu Met Gly Thr Leu
20 25 30
Gly Ile Val Ala Ala Tyr Thr Leu Gly Ile Val Cys Pro Ile
35 40 45
<210>15
<211>22
<212>PRT
<213>未知
<220>
<223>二肽
<400>15
Thr Ile His Asp Ile Ile Leu Glu Cys Val Lys Lys Pro Leu Leu Met
1 5 10 15
Gly Thr Leu Gly Ile Val
20
<210>16
<211>21
<212>PRT
<213>未知
<220>
<223>二肽
<400>16
Thr Leu Gly Ile Val Cys Pro Ile Lys Lys Pro Tyr Met Leu Asp Leu
1 5 10 15
Gln Pro Glu Thr Thr
20
<210>17
<211>22
<212>PRT
<213>未知
<220>
<223>PADRE与mK9M p53表位连接
<220>
<221>misc_feature
<222>(3)..(3)
<223>Xaa=环己基丙氨酰
<400>17
Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Lys Tyr Ile
1 5 10 15
Cys Asn Ser Ser Cys Met
20
<210>18
<211>9
<212>PRT
<213>未知
<220>
<223>Mart-1/Melan-A
<400>18
Ala Ala Gly Ile Gly Ile Leu Thr Val
1 5
<210>19
<211>10
<212>PRT
<213>未知
<220>
<223>Mart-1/Melan-A
<400>19
Glu Ala Ala Gly Ile Gly Ile Leu Thr Val
1 5 10
<210>20
<211>9
<212>PRT
<213>未知
<220>
<223>Mart-1/Melan-A
<400>20
Ile Leu Thr Val Ile Leu Gly Val Leu
1 5
<210>21
<211>10
<212>PRT
<213>未知
<220>
<223>Mart-1/Melan-A
<400>21
Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu
1 5 10
<210>22
<211>11
<212>PRT
<213>未知
<220>
<223>Mart-1/Melan-A
<400>22
Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu Thr
1 5 10
<210>23
<211>9
<212>PRT
<213>未知
<220>
<223>MCIR
<400>23
Thr Ile Leu Leu Gly Ile Phe Phe Leu
1 5
<210>24
<211>9
<212>PRT
<213>未知
<220>
<223>MCIR
<400>24
Phe Leu Ala Leu Ile Ile Cys Asn Ala
1 5
<210>25
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>25
Lys Thr Trp Gly Gln Tyr Trp Gln Val
1 5
<210>26
<211>10
<212>PRT
<213>未知
<220>
<223>Gp100
<400>26
Ala Met Leu Gly Thr His Thr Met Glu Val
1 5 10
<210>27
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>27
Met Leu Gly Thr His Thr Met Glu Val
1 5
<210>28
<211>10
<212>PRT
<213>未知
<220>
<223>Gp100
<400>28
Ser Leu Ala Asp Thr Asn Ser Leu Ala Val
1 5 10
<210>29
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>29
Ile Thr Asp Gln Val Pro Phe Ser Val
1 5
<210>30
<211>10
<212>PRT
<213>未知
<220>
<223>Gp100
<400>30
Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu
1 5 10
<210>31
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>31
Tyr Leu Glu Pro Gly Pro Val Thr Ala
1 5
<210>32
<211>10
<212>PRT
<213>未知
<220>
<223>Gp100
<400>32
Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val
1 5 10
<210>33
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>33
Arg Leu Pro Arg Ile Phe Cys Ser Cys
1 5
<210>34
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>34
Leu Ile Tyr Arg Arg Arg Leu Met Lys
1 5
<210>35
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>35
Ala Leu Asn Phe Pro Gly Ser Gln Lys
1 5
<210>36
<211>10
<212>PRT
<213>未知
<220>
<223>Gp100
<400>36
Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys
1 5 10
<210>37
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>37
Ala Leu Leu Ala Val Gly Ala Thr Lys
1 5
<210>38
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>38
Ala Leu Leu Ala Val Gly Ala Thr Lys
1 5
<210>39
<211>9
<212>PRT
<213>未知
<220>
<223>Gp100
<400>39
Val Tyr Phe Phe Leu Pro Asp His Leu
1 5
<210>40
<211>8
<212>PRT
<213>未知
<220>
<223>Gp100
<400>40
Ser Asn Asp Gly Pro Thr Leu Ile
1 5
<210>41
<211>10
<212>PRT
<213>未知
<220>
<223>PSA
<400>41
Val Ser His Ser Phe Pro His Pro Leu Tyr
1 5 10
<210>42
<211>10
<212>PRT
<213>未知
<220>
<223>PSA
<400>42
Phe Leu Thr Pro Lys Lys Leu Gln Cys Val
1 5 10
<210>43
<211>10
<212>PRT
<213>未知
<220>
<223>PSA
<400>43
Val Ile Ser Asn Asp Val Cys Ala Gln Val
1 5 10
<210>44
<211>10
<212>PRT
<213>未知
<220>
<223>PSM
<400>44
His Ser Thr Asn Gly Val Thr Arg Ile Tyr
1 5 10
<210>45
<211>9
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>45
Lys Cys Asp Ile Cys Thr Asp Glu Tyr
1 5
<210>46
<211>11
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>46
Ser Ser Asp Tyr Val Ile Pro Ile Gly Thr Tyr
1 5 10
<210>47
<211>9
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>47
Tyr Met Asp Gly Thr Met Ser Gln Val
1 5
<210>48
<211>9
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>48
Met Leu Leu Ala Val Leu Tyr Cys Leu
1 5
<210>49
<211>9
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>49
Ala Phe Leu Pro Trp His Arg Leu Phe
1 5
<210>50
<211>9
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>50
Ser Glu Ile Trp Arg Asp Ile Asp Phe
1 5
<210>51
<211>9
<212>PRT
<213>未知
<220>
<223>酪氨酸酶
<400>51
Met Ser Leu Gln Arg Gln Phe Leu Arg
1 5
<210>52
<211>9
<212>PRT
<213>未知
<220>
<223>TRP1
<400>52
Ser Val Tyr Asp Phe Phe Val Trp Leu
1 5
<210>53
<211>9
<212>PRT
<213>未知
<220>
<223>TRP2
<400>53
Thr Leu Asp Ser Gln Val Met Ser Leu
1 5
<210>54
<211>9
<212>PRT
<213>未知
<220>
<223>TRP2
<400>54
Leu Leu Gly Pro Gly Arg Pro Tyr Arg
1 5
<210>55
<211>9
<212>PRT
<213>未知
<220>
<223>p53
<400>55
Ala Asn Asp Pro Ile Phe Val Val Leu
1 5
Claims (55)
1.一种组合物,包括:
载体,其包含疏水物质的连续相;
脂质体;和
至少一种抗原,其能够诱导细胞毒性T淋巴细胞(CTL)反应。
2.根据权利要求1所述的组合物,进一步包括至少一种T辅助细胞表位。
3.根据权利要求2所述的组合物,其中所述T辅助细胞表位是与所述至少一种抗原相分离的分子。
4.根据权利要求2所述的组合物,其中所述抗原包含所述T辅助细胞表位。
5.根据权利要求1所述的组合物,其中所述抗原为多肽。
6.根据权利要求1所述的组合物,其中所述抗原包含一个CTL表位或多个CTL表位的组合。
7.根据权利要求6所述的组合物,其中所述CTL表位来源于病毒。
8.根据权利要求7所述的组合物,其中所述CTL表位来源于人乳头状瘤病毒。
9.根据权利要求8所述的组合物,其中所述CTL表位为人乳头状瘤病毒(HPV)的E6或E7蛋白的表位。
10.根据权利要求9所述的组合物,其中所述E6表位包含肽序列TIHDIILECV(T10V;SEQ ID NO:5)。
11.根据权利要求9所述的组合物,其中所述E7表位包含选自由下列序列组成的组中的肽序列:RAHYNIVTF(R9F;SEQ ID NO:1)、YMLDLQPETT(Y10T;SEQ ID NO:2)、LLMGTLGIV(L9V;SEQ ID NO:3)和TLGIVCPI(T81;SEQ ID NO:4)。
12.根据权利要求8所述的组合物,其中所述CTL表位为HPV的E6和E7蛋白的CTL表位的混合物。
13.根据权利要求12所述的组合物,其中所述表位的混合物包含Y10T(SEQ ID NO:2)、L9V(SEQ ID NO:3)、T81(SEQ ID NO:4)和T10V(SEQ IDNO:5)肽序列。
14.根据权利要求13所述的组合物,其中所述表位被连接到一起形成一个肽(AB2)(SEQ ID NO:14)。
15.根据权利要求6所述的组合物,包含连接起来形成单个多肽的多个CTL表位。
16.根据权利要求6所述的组合物,其中所述CTL表位来源于肿瘤相关蛋白。
17.根据权利要求16所述的组合物,其中所述肿瘤相关蛋白为p53。
18.根据权利要求16所述的组合物,其中所述肿瘤相关蛋白为酪氨酸酶相关蛋白-2(TRP-2)。
19.根据权利要求6所述的组合物,其中所述抗原包含p53表位和TRP-2表位的混合物。
20.根据权利要求17所述的组合物,其中所述p53表位包含肽序列KYICNSSCM(mK9M)(SEQ ID NO:8)。
21.根据权利要求18所述的组合物,其中所述TRP-2表位包含肽序列SVYDFFVWL(SEQ ID NO:7)。
22.根据权利要求18所述的组合物,其中所述TRP-2表位包含肽序列VYDFFVWL(SEQ ID NO:6)。
23.根据权利要求2所述的组合物,其中所述T辅助细胞表位为通用T辅助细胞表位。
24.根据权利要求23所述的组合物,其中所述T辅助细胞表位为PADRE(pan-DR表位)(SEQ ID NO:10)。
25.根据权利要求2所述的组合物,其中所述T辅助细胞表位为F21E(SEQ ID NO:11)。
26.根据权利要求2所述的组合物,其中所述T辅助细胞表位被融合到所述至少一种抗原。
27.根据权利要求26所述的组合物,其中所述T辅助细胞表位为PADRE并且至少一种抗原是CTL表位。
28.根据权利要求1所述的组合物,进一步包括佐剂。
29.根据权利要求28所述的组合物,其中所述佐剂为CpG寡聚脱氧核苷酸(CpG ODN)(SEQ ID NO:12)。
30.根据权利要求28所述的组合物,其中所述佐剂为脂肽。
31.根据权利要求30所述的组合物,其中所述脂肽为Pam3Cys-SKKKK。
32.根据权利要求1~31中任一项所述的组合物在制备用于控制肿瘤大小的药物中的应用。
33.根据权利要求1~31中任一项所述的组合物在制备用于在被治疗者中治疗癌症或抑制或防止癌细胞生长或增殖的药物中的应用。
34.根据权利要求33所述的应用,其中所述癌症选自由宫颈癌、外阴癌、黑色素瘤、乳腺癌、肺癌、卵巢癌、多发性骨髓瘤、B细胞淋巴瘤、肝细胞瘤、肉瘤、膀胱癌、前列腺癌、甲状腺肿瘤、头颈肿瘤、结肠癌、直肠癌、肾癌、胰腺癌、胃癌、腺癌、T细胞白血病、淋巴肉瘤、子宫癌、食道癌、非何杰金氏淋巴瘤、子宫内膜癌和RCC肿瘤所组成的组中。
35.一种用于在被治疗者中治疗癌症的试剂盒,包括权利要求1~30中任一项所述的组合物及其使用说明书。
36.权利要求33或34所述的应用,其中所述被治疗者为哺乳动物。
37.根据权利要求36所述的应用,其中所述哺乳动物为人。
38.根据权利要求1~31中任一项中所述的组合物,其中所述脂质体包含磷脂。
39.根据权利要求38所述的组合物,其中所述磷脂具有至少一种选自磷酸甘油、磷酸乙醇胺、磷酸丝氨酸、磷酸胆碱和磷酸肌醇中的头部基团。
40.根据权利要求1所述的组合物,其中所述脂质体包含未酯化的胆固醇。
41.根据权利要求1所述的组合物,其中所述脂质体包含卵磷酯Phospholipon90G中的脂类。
42.根据权利要求1所述的组合物,其中所述脂质体从原始细菌得到。
43.根据权利要求1所述的组合物,其中所述脂质体从天然脂类、合成的脂类、神经鞘脂类、醚脂类、甾醇类、心磷脂、阳离子脂类或聚乙二醇或其他聚合物修饰的脂类得到。
44.根据权利要求43所述的组合物,其中所述合成的脂类包括选自月桂酰、肉豆蔻酰、棕榈酰、硬酯酰、二十烷酰、油酰、亚油酰和伊鲁克的脂肪酸构成成分或其组合。
45.一种组合物,所述组合物用包括将脂质体和能够诱导细胞毒性T淋巴细胞(CTL)反应的抗原与含有疏水物质的连续相的载体结合的方法制备。
46.根据权利要求45所述的组合物,其中所述方法进一步包括将T辅助细胞表位与所述脂质体、所述抗原和所述载体结合。
47.根据权利要求45或46所述的组合物,其中所述方法进一步包括将佐剂与所述脂质体、所述抗原和所述载体结合。
48.根据权利要求45所述的组合物,其中所述抗原在所述脂质体中被胶囊化。
49.根据权利要求46所述的组合物,其中所述T辅助细胞表位在所述脂质体中胶囊化。
50.根据权利要求47所述的组合物,其中所述佐剂在所述脂质体中胶囊化。
51.根据权利要求45所述的组合物,其中所述脂质体在与所述载体结合前冻干。
52.根据权利要求46所述的组合物,其中所述T辅助细胞表位是与所述抗原相分离的分子。
53.根据权利要求46所述的组合物,其中所述抗原包含所述T辅助细胞表位。
54.根据权利要求45所述的组合物,其中所述抗原是在权利要求6~22任一项中限定的抗原。
55.根据权利要求46所述的组合物,其中所述T辅助细胞表位是在权利要求23~27任一项中限定的T辅助细胞表位。
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CA2523032 | 2005-10-07 | ||
CA002523032A CA2523032A1 (en) | 2005-10-07 | 2005-10-07 | Vaccines for cancer therapy |
CA002533705A CA2533705A1 (en) | 2005-10-07 | 2006-01-13 | Use of liposomes in a water-in-oil emulsion or in a continuous hydrophobic carrier as a vehicle for cancer treatment |
CA2533705 | 2006-01-13 | ||
CA2542212 | 2006-04-07 | ||
CA002542212A CA2542212A1 (en) | 2005-10-07 | 2006-04-07 | Use of liposomes in a water-in-oil emulsion or in a continuous hydrophobic carrier as a vehicle for cancer treatment |
US80657306P | 2006-07-05 | 2006-07-05 | |
US60/806,573 | 2006-07-05 | ||
PCT/CA2006/001640 WO2007041832A1 (en) | 2005-10-07 | 2006-10-05 | Use of liposomes in a carrier comprising a continuous hydrophobic phase as a vehicle for cancer treatment |
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EP1948225A4 (en) | 2008-12-24 |
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CA2622464A1 (en) | 2007-04-19 |
US10272042B2 (en) | 2019-04-30 |
AU2006301891B2 (en) | 2012-09-06 |
CN101282742A (zh) | 2008-10-08 |
ES2451599T3 (es) | 2014-03-27 |
JP5528703B2 (ja) | 2014-06-25 |
US20150202152A1 (en) | 2015-07-23 |
EP1948225A1 (en) | 2008-07-30 |
US9925142B2 (en) | 2018-03-27 |
CA2542212A1 (en) | 2007-04-07 |
CA2622464C (en) | 2019-09-17 |
WO2007041832A1 (en) | 2007-04-19 |
CA2523032A1 (en) | 2007-04-07 |
EP1948225B1 (en) | 2013-12-11 |
CA2533705A1 (en) | 2007-07-13 |
AU2006301891A1 (en) | 2007-04-19 |
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