CN104837497B - 用于预防和治疗HPV-相关的肿瘤和其它表达p16的肿瘤的p16INK4a衍生肽 - Google Patents
用于预防和治疗HPV-相关的肿瘤和其它表达p16的肿瘤的p16INK4a衍生肽 Download PDFInfo
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Abstract
描述了能够增加T细胞的IFN‑γ分泌或诱导T细胞增殖的细胞周期蛋白依赖性激酶抑制剂p16的特定片段以及所述片段用于免疫针对HPV‑相关的或其它表达p16INK4a的癌、优选为晚期癌的个体的用途。
Description
技术领域
本发明涉及细胞周期蛋白依赖性激酶抑制剂p16INK4a的特定片段以及所述片段用于免疫针对表达p16INK4a的肿瘤的个体的用途。
背景技术
每年全世界有几百万人罹患癌，并且死于癌。尽管强化治疗研究，但这些死亡率已经多年保持未变。迄今为止，患有癌的患者经常不得不经历癌去除手术或化学治疗或放射治疗。然而，这伴随着非常巨大的副作用，所述副作用然后促成患有癌的患者的死亡率。有趣地，人乳头瘤病毒(HPV)与所有癌症中超过5％的癌症的形成相关(Parkin和Bray，2006)。预防性HPV接种已经为可用的，但在已经被感染的人中未示出治疗效果(Hildesheim等人，2007)。因此，存在对新型治疗方案的需要。
发明内容
因此，本发明的目的为提供用于治疗和预防HPV-相关的肿瘤和其它表达 p16INK4a的肿瘤的手段。
根据本发明，这通过权利要求中限定的主题来实现。源自观察产生本发明的研究：当由于负反馈环被HPV致癌基因产物E7破坏而细胞实现转化的且可能为恶性表型时，细胞蛋白p16INK4a在HPV感染的细胞中表达(Sano等人，1998； Klaes等人，2001)。因此，p16INK4a在几乎所有HPV-诱导的癌和高度瘤形成前，包括宫颈肿瘤、外阴肿瘤、阴道肿瘤、阴茎肿瘤、肛门肿瘤以及头颈肿瘤中强烈表达(Ishikawa等人，2006；Samama等人，2006；Missaoui等人，2006；Santos 等人，2006；Roma等人，2008；Hafkamp等人,2003)。在生理条件下，p16INK4a仅在经历随后衰老的细胞中表达，因此，几乎未在正常组织中发现表达(Beauséjour等人，2007)。除HPV-致癌基因驱使HPV-相关的肿瘤中的p16INK4a表达之外，也发现p16INK4a在与HPV感染无关的各种肿瘤或在已经发现HPV但未证明病毒致癌作用的肿瘤中过表达，其包括一部分黑素瘤和非黑素瘤皮肤癌 (Nindl等人，2004；Busch等人，2010)、肺癌(Leversha等人，2003；Esposito 等人，2004)、食道癌、胃癌和结肠直肠癌(Ding等人，2010；Kim等人，2005)以及肾癌、膀胱癌、卵巢癌、子宫内膜癌和乳腺癌(Ikuerowo等人2007；Buza 等人，2010；Giordano等人，2008；Giordano等人，2007；di Vinci等人，2005)。已知视网膜母细胞瘤抑制基因的突变导致p16INK4a的表达上调(Okamoto等人， 1994)。然而，在这些情况下强的p16INK4a表达的潜在机制最可能是更异种的并且最终不能被理解。
很久以来，癌细胞中内源性基因产物的过表达已被认为是肿瘤相关抗原的有价值的来源。针对此类抗原的免疫响应已经在各种癌症患者中观察到并且已经在免疫治疗试验中有所增加(等人，2003；Finn，2008；Rescigno等人， 2007)。
在形成本发明的实验期间，可证明从健康个体的外周血样品分离的T淋巴细胞可在体外用p16INK4a衍生肽特异性刺激以及来自宫颈癌患者的CD4+和 CD8+T细胞对相同的p16INK4a肽示出自发反应性并且产生细胞毒性T细胞系和宫颈癌细胞系，所述细胞毒性T细胞系能够攻击并杀死共培养的HLA匹配的负载有p16INK4a的细胞。换言之，p16INK4a的片段具有高度免疫原性，从而诱导针对 p16INK4a的非常强的免疫响应。此外，已经证明也可检测针对p16INK4a的体液免疫响应。
p16INK4a的所述表达模式以及针对p16INK4a的与任何自身免疫疾病不相关的自发免疫响应的发现使p16INK4a成为用于免疫具有表达p16INK4a的癌症的患者的有前景的候选者。针对p16INK4a的活性诱导的强免疫响应可特异性破坏HPV转化的细胞和其它表达p16INK4a的癌细胞。具有这些p16INK4a肽的供体T细胞的接种在细胞培养实验中进行。使用p16INK4a肽的另外的实验揭示宫颈癌患者中的自发的 T细胞响应，证实特定的p16INK4a肽在体内也为免疫原性的。因此，使用这些肽基于免疫的癌症的预防或治疗应对患者具有若干益处。p16INK4a在所有HPV-相关的癌症(不论HPV类型)以及各种其它癌症类型中强烈表达。p16INK4a免疫的严重的副作用是未预期的，因为p16INK4a在正常组织中几乎不表达并且在具有针对 p16INK4a的自发的免疫响应的个体中未观察到自身免疫现象。最后，由于抗原损失的免疫逃避是非常不同的，因为p16INK4a表达与肿瘤细胞的恶性表型复杂地相关。
附图说明
图1：用p16INK4a肽刺激供体T细胞之前(A)和之后(B)的ELISpot(干扰素γ)结果
斑点计数通过减去在不含肽的孔中的背景斑点检测而标准化。在B中用肽p16INK4a_37-63、p16INK4a_51-80和p16INK4a_73-104刺激的细胞中增加的斑点计数与第0天相比变得清晰。CEF＝CMV，EBV，流感(flu)肽混合物阳性对照。
图2：在针对阳性对照病毒混合物(CEF)以及针对七条30聚体p16INK4a肽的23名患者 (Tx和Fx)和15名健康对照(BCx)中的ELISpot(干扰素γ)结果(表1)
结果为调整后的背景并且仅考虑了截断值之上的斑点(阴性对照孔中的两倍斑点+ 针对各个p16INK4a肽的反应性的两个标准差)。两名个体具有CD4+响应，剩余的个体具有CD8+响应。na＝未分析的。
图3：铬释放测定
来自宫颈癌患者的CD8T细胞(HLA A2 A3 B7 B15 Cw3 Cw7)诱导HLA匹配的负载有p16INK4a_37-63的B细胞(黑方块)裂解但不诱导不含p16INK4a肽的相同B细胞裂解(空心三角形)。宫颈癌细胞系HeLa(p16INK4a+、HLA A68、 B15、B95、Cw7 Cwl2)和Caski(p16INK4a+、HLAA2、A3、B7、B37、Cw5、 Cw7)裂解同时未检测到ME180和K562裂解。
图4：在用七种p16INK4a肽(表1)刺激之后来自具有宫颈非典型增生的妇女的外周
血单核细胞的增殖
示出了在用p16INK4a肽、阳性对照(促分裂原PHA和破伤风类毒素)和阴性对照(无抗原)孵育PBMC之后进行的BrdU增殖测定的光密度。虚线指示阳性响应的截断值。星号指示诱导增殖的p16INK4a肽。示出了来自三名发生反应的患者以及一名阴性患者的结果。
图5：蛋白质印迹中的血清学p16INK4a反应性
(a)(1)纯化的His-标签的p16INK4a的银染色。(2)具有单克隆p16INK4a抗体 E6H4的纯化的His-标签的p16INK4a的蛋白质印迹。
(b)示出了6个代表性阳性血清。蛋白大小对应于单克隆抗体(E6H4)针对重组蛋白的反应。
(c)阴性血清的实例(1和2)。
(d)在血清与重组p16INK4a预孵育之前(1)和之后的(2)p16INK4a血清反应性。
具体实施方式
因此，本发明涉及能够诱导针对p16INK4a的免疫响应的细胞周期蛋白依赖性激酶抑制剂p16Ink4a的特定片段。免疫响应被定义为满足以下标准中的至少一种的状态：1.CD8-阳性T细胞的诱导，如通过细胞毒性测定或IFN-γ分泌或穿孔素表达或颗粒酶B表达或可以由CD8-阳性T细胞产生的其它细胞因子可检测的，通过ELISpot或细胞内细胞因子染色或细胞因子ELISA或等同方法如上背景可测量的。2.CD4-阳性T细胞的诱导，如通过细胞因子分泌可检测的，通过 ELISpot或细胞内细胞因子染色或细胞因子ELISA或等价方法如上背景可测量的。细胞因子可以包括IFN-α、IFN-γ、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、 IL-13、IL-17、TNF-α、TGF-β或可以由CD4-阳性T细胞产生的其它细胞因子。 3.抗体的诱导，如通过蛋白印记、ELISA以及等同或相关方法可检测的。4.未由如1和2中所述的CD8-阳性或CD4-阳性T细胞介导的任何种类的细胞免疫响应的诱导。
p16INK4a的片段由以下组成：
(a)下述氨基酸序列中的任一个：
(a1)MEPAAGSSMEPSADWLATAAARGRV；
(a2)TAAARGRVEEVRALLEAGALPNAPNSY；
(a3)LPNAPNSYGRRPIQVMMMGSARVAELL；
(a4)VMMMGSARVAELLLLHGAEPNCADPATLTR；
(a5)ADPATLTRPVHDAAREGFLDTLWLHRAGARL；
(a6)HRAGARLDVRDAWGRLPVDLAEELGHRDVAR；
(a7)GHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD
(b)的片段的功能等同物，其仍能够诱导针对p16INK4a的免疫响应；或
(c)(a)和/或(b)的片段的组合。
例如，本文使用的术语“功能等同物”涉及，(a)的变体或片段，其仍能够诱导针对p16INK4a的免疫响应，因此，仍可用作有效的疫苗。变体的特征在于氨基酸缺失、取代和/或添加。优选地，氨基酸差异是由于一种或多种保守氨基酸取代。术语“保守氨基酸取代”涉及脂族或疏水性氨基酸Ala、Val、Leu和Ile 的替换；羟基残基Ser和Thr的替换；酸性残基Asp和Glu的替换；酰胺残基 Asn和Gln的替换，碱性残基Lys、Arg和His的替换；芳族残基Phe、Tyr和 Trp的替换以及小型氨基酸Ala、Ser、Thr、Met和Gly的替换。
对于示出特定程度的同一性的肽的制备，如，基因工程可用于在克隆的DNA 序列的特定位置处引入氨基酸变化以鉴定对于肽功能重要的区域。例如，可使用定点诱变或丙氨酸扫描诱变(在分子的每个残基处引入单个丙氨酸突变) (Cunningham和Wells，1989)。然后，使用实施例中的测定，所得的突变分子可用于测试免疫原性。
优选地，变体的特征在于不超过8个氨基酸，更优选为不超过6个氨基酸，甚至更优选地，不超过4个氨基酸取代、缺失和/或添加。
在原始的p16Ink4a-片段的片段中，特定氨基酸序列的至少5个连续氨基酸、优选至少10个连续氨基酸、更优选至少15个连续氨基酸、甚至更优选至少20 个连续氨基酸被剩下。此类片段仍能够诱导针对p16INK4a的免疫响应，并且因此，仍可用作有效的疫苗。
本发明还提供了编码本发明的片段的核酸或含有此类核酸的载体。将遗传物质直接注入活的宿主引起其少量的细胞产生引入的基因产物。宿主内的不适当的基因表达具有重要的免疫学影响，从而导致针对基因递送的抗原的宿主的特定的免疫活化。裸质粒DNA的直接注入引起对基因疫苗编码的抗原强的免疫响应。一旦注入质粒DNA构建体，宿主细胞摄取外源DNA，从而在细胞内部表达病毒基因并产生p16INK4a。这种形式的抗原呈递和处理诱导MHC以及I类和II类限制的细胞和体液免疫响应。DNA疫苗由通常含有两个单元的载体组成：抗原表达单元，其由启动子/增强子序列，随后编码抗原(FSP)和多腺苷酸化序列组成以及由对于载体扩增和选择必需的序列组成的制备单元。具有疫苗插入物的载体的构建使用重组DNA技术来实现并且本领域技术人员知道可用于该方法的载体。DNA免疫的效率可通过使DNA对降解保持稳定以及提高DNA至抗原呈递细胞递送的效率来改善。这已经通过用DNA包覆生物可降解的阳离子微粒(诸如用十六烷基三甲基溴化铵配制的聚(丙交酯-共-乙交酯))来证明。此类DNA包覆的微粒在升高CTL上与重组牛痘病毒一样有效率，特别是当与明矾混合时。直径为300nm的粒子对于通过抗原呈递细胞的摄取看起来为最有效的。
可以利用多种表达载体，如，质粒或病毒载体以含有且表达编码本发明的片段的核酸序列。
优选的病毒载体为痘病毒、腺病毒、逆转录病毒、疱疹病毒或腺相关病毒 (AAV)。特别优选的痘病毒为牛痘病毒、NYVAC、鸟痘病毒、金丝雀痘病毒、 ALVAC、ALVAC(2)禽痘病毒或TROVAC。
重组的基于甲病毒属的载体已经用于改善DNA接种效率。将编码本发明的片段的基因插入甲病毒属复制子，替换结构基因，而保持非结构复制酶基因完整。辛德毕斯病毒(Sindbis virus)和塞姆利基森林病毒(Semliki Forest virus) 已经用于构造重组甲病毒属复制子。然而，不像常规的DNA接种，甲病毒属载体仅瞬时表达。由于由该载体表达的高水平的蛋白、复制子诱导的细胞因子响应或复制子诱导的凋亡导致树突细胞增强抗原摄取，所以甲病毒属复制子提高免疫响应。
本发明还提供了含有适合于个体免疫的量的本发明的片段、核酸序列或载体以及优选一种或多种常见助剂的药物组合物。此类片段、核酸序列或载体本身可存在或与载体的组合存在。个体中的载体非免疫原性的是有利的。此类载体可以为个体自己的蛋白或外源蛋白或其片段。载体，诸如血清白蛋白、纤维蛋白原或转铁蛋白或其片段为优选的。片段含有表位，所述表位被细胞毒性T 细胞，如CD8+T细胞或CD4T细胞识别，并且可以诱导免疫响应。细胞循环调控蛋白的此类表位可通过本领域技术人员熟悉的方法确定。各种片段同时存在也可以为有利的。对于以上片段的重组制备，参见，如，Sambrook等人，MolecularCloning:A Laboratory Manual，第2版，冷泉港实验室出版社，冷泉港，纽约 (1989)。
本发明还涉及本发明的片段、核酸序列或载体用于制备预防或治疗表达 p16INK4a的瘤形成前、瘤形成或癌(包括晚期癌)的疫苗的用途。
例如，这些可以为HPV诱导的、表达p16INK4a的阴肛部癌，特别是宫颈癌或头颈癌以及非HPV诱导的表达p16INK4a的肿瘤。同样，良性修饰诸如乳头瘤、腺瘤、增生或类似的上皮增殖、间质或造血增殖也计数在当中。
采用的术语“个体”包括任何种类的并且能够患上癌的个体。此类个体的实例为人和动物以及其细胞。
采用的术语“适合于个体免疫的量”包含以上解释相应地应用于并且可使个体免疫的本发明的片段的任何量。例如，所述量取决于免疫是否旨在预防性或治疗性治疗。另外，个体的年龄、性别和体重对于确定所述量起作用。通过注射给予个体100μg至1g的p16片段是有利的。可以在个体的各个位置进行肌内、皮下、皮内或以任何其它施用形式注射。进行一种或多种具有约等量的“加强注射”也可为有利的。在这种情况下，使用个体注射的各自细胞循环调控蛋白的不同片段可为特别有利的。
采用的术语“常见助剂”包括适合于免疫个体的药物组合物的任何助剂。此类助剂为，如，免疫佐剂，诸如GM-CSF或弗氏佐剂(Freund's adjuvant)、缓冲的常见盐溶液、水、乳剂诸如油/水乳剂、润湿剂、无菌溶液等。
通过本发明来使个体免疫，特别是人和动物是可能的。免疫通过抗体的诱导以及T细胞的刺激两者而发生。因此，采取针对瘤形成前、瘤形成和癌的预防性和治疗性步骤是可能的。
本发明通过以下实施例来解释。
实施例1
在具有HPV-相关的瘤形成的患者中针对p16INK4a肽的T细胞反应性
为了评价具有HPV-相关的肿瘤的患者是否提高强烈过表达的p16INK4a的T 细胞以及针对强烈过表达的p16INK4a的T细胞响应至何种程度，应用了允许详细表征针对p16INK4a抗原的免疫响应的不同的方法。在宫颈癌患者中针对p16INK4a的自发免疫响应的发现证明通常抗原以及特定的p16lNK4a片段的免疫原性并为免疫含有p16INK4a片段的具有表达p16INK4a的肿瘤的患者提供了合理性。
将来自13名具有表达p16INK4a的高度宫颈非典型增生(CIN2/3)的妇女的外周血单核细胞(PBMC)与p16INK4a肽(表1)孵育以通过应用作为用p16INK4a肽挑战之后的淋巴组织增生的潜能的总体测量的BrdU测定来确定免疫细胞的增殖能力。BrdU测定为基于DNA合成期间胸苷类似物5-溴-2’-脱氧尿苷并入的测量应用于细胞增殖的定量的比色免疫测定。将在补充有10％人血清的IMDM 培养基中的PBMC以150.000个细胞/50μl/孔的密度接种在96孔微量滴定板(平底)中。在存在七条p16INK4a肽(表1)、破伤风类毒素(20ng/ml，Calbiochem，La Jolla，CA)以及促分裂原PHA-L(5μg/ml，Roche，曼海姆，德国)(作为阳性对照)和不含抗原(作为阴性对照)的情况下将在每4个重复孔中的细胞在37℃、5％CO2下孵育6天。在第6天时，将10μl/孔的于IMDM培养基+10％的人血清中以1:100稀释的BrdU标记的溶液(使用来自Roche，曼海姆，德国的细胞增殖ELISA，BrdU(比色)的所有BrdU测定试剂)加入至每个孔中并在37℃，5％CO2下再孵育18个小时。在第7天，将板在1200rpm下离心分离10min，去除50μl上清液并转移至新的V底板中以最终储存在-80℃下用于细胞因子分析。将剩余的细胞通过使用电吹风干燥约15分钟并将150μl/孔的 FixDenat溶液加入至细胞并在室温下孵育30分钟，然后通过小心地弹去和轻拍去除FixDenat。加入100μl/孔的抗-BrdU-POD工作液并在室温孵育90分钟，然后去除并用100μl TMB基质替换，所述基质在室温孵育30分钟。通过加入25μl/ 孔的1N H2SO4终止酶反应并在450nm(参考波长为620nm)测量光密度(OD)。阳性反应的截断值设置为不含抗原的阴性对照中的OD的标准差的三倍。来自 13名测试的妇女的其中3名的PBMC示出响应于p16INK4a肽的增殖，这指示与肽的孵育具有活化的增殖性记忆T细胞响应。总之，诱导肽的响应模式为异种的，这指示p16抗原内的各种T细胞表位。一名患者示出对肽p16INK4a_18-44、 p16INK4a_37-63、p16INK4a_73-107和p16INK4a_123-156的响应，一名患者示出对肽p16INK4a_51-80和p16INK4a_98-128的响应，以及一名患者示出对肽p16INK4a_1-25和p16INK4a_37-63的响应(图4)。
为了证明特定的p16片段能够诱导作为Thl响应的一种迹象的干扰素γ分泌，来自23名具有浸润性宫颈癌和强烈的p16INK4a过表达的高度癌前期病变 (CIN2/3)的患者的T细胞在干扰素γELISpot测定中针对七条p16INK4a肽(表 1)进行测试。使用Ficoll离心分离、塑性粘附以及抗体偶联磁珠(CD11，CD16， CD19，CD36，CD56，Pan T细胞分离试剂盒，Milteny，贝尔吉施格拉德巴赫，德国)由肝素化血液分离T细胞。通过将塑性粘附细胞与IL4和GM-SCF(每个为1000U/ml)培养7天生成树突细胞并将所述树突细胞用作ELIspot中的抗原呈递细胞。在与由2x 104个树突细胞提供的各个肽进行短的(2至5天)体外预敏化之后，测试每个105个T细胞。
当减去背景(阴性对照孔中的2倍斑点+针对各个p16INK4a肽的反应性的2个标准差)时，可鉴定针对p16INK4a_37-63肽反应的7名宫颈癌患者中的T细胞 (CD4或CD8)(图2)。
实施例2
用p16INK4a肽体外引发健康供体T细胞
测试覆盖整个p16INK4a氨基酸序列的七条长的25-35聚体肽(每个具有7-13 个氨基酸重叠)以定义能够诱导健康供体体外分泌干扰素γ的T细胞的p16INK4a片段(表1)。
表1在体外实验中使用的七条重叠的p16INK4a肽
为了示出p16INK4a片段可刺激健康供体T细胞体外分泌干扰素γ和鉴定大多数免疫原性p16INK4a来源的表位，研究分离自健康供体的外周血的T细胞是否可被这些p16INK4a肽体外刺激。当在所谓的ELISpot实验中用各个p16INK4a肽挑战时，如果p16INK4a肽能够在细胞培养实验中诱导特异性T细胞响应，则T细胞分泌细胞因子。在ELISpot测定中，细胞因子(干扰素γ)可通过具有随后颜色反应的特异性抗体来检测。
通过Ficoll Plaque梯度密度离心分离由一名健康供体的肝素化血液(100ml) 分离外周血单核细胞(PBMC)。将5至10x107个PBMC通过塑性粘附和抗体偶联的磁珠(CD11，CD16，CD19，CD36，CD56，Pan T细胞分离试剂盒，Milteny，贝尔吉施格拉德巴赫，德国)分离成单核细胞和T细胞。将单核细胞与GM-CSF 和IL-4(每个为1000U/ml)培养7天以生成抗原呈递树突细胞。
将2x107个T细胞与2x106个先前用p16INK4a肽(10pg/ml)脉冲4小时的树突细胞孵育4小时以实现抗原的呈递。对于7条p16INK4a肽中的每一者，进行单独的刺激方法。
在5周时段内每7天将T细胞用p16INK4a肽脉冲的树突细胞再刺激并用IL-2 和IL-7(10U/ml)处理。
在刺激前(第0天)和最后刺激之后(第35天)在干扰素γELISpot测定中测量p16INK4a肽特异性的T细胞响应。对于ELISpot测定，将96孔硝酸纤维素板(MAHAN4550Millipore)用浓度为0.75ug/孔的抗扰素γ抗体1-D1K (Mabtech，Nacka Strand，瑞典)涂布。将每个105个T细胞用2x104个树突细胞呈递的各个肽测试。在37℃孵育12小时之后，分泌的干扰素γ的检测通过用生物素化的第二抗干扰素γ抗体7BG-1(Mabtech)、链霉亲和素-碱性磷酸酶缀合物和BCIP/NBT基质溶液(Sigma Aldrich，St.Louis，USA)检测来实现。
虽然在第0天未检测到针对七条p16INK4a肽的任一者的反应性，但仅检测到针对用作阳性对照的病毒肽混合物(CEF＝CMV&EBV&流感)的反应性，在第35天时，当针对用各个p16INK4a肽脉冲的靶细胞测试时，用肽p16INK4a_37-63、 p16INK4a_51-80和p16INK4a_73-104刺激的T细胞在ELISpot中示出增加的干扰素γ分泌，而当针对用剩余的p16INK4a肽(图1)脉冲的细胞测试时，用肽p16INK4a_37-63、p16INK4a_51-80和p16INK4a_73-104刺激的T细胞在 ELISpot中未示出增加的干扰素γ分泌。
实施例3
p16INK4a反应性T细胞的宫颈癌细胞系的裂解
使用不同的宫颈癌细胞系以及HLA匹配的负载有p16INK4a肽的B细胞作为靶标，活化的T细胞裂解表达p16INK4a的宫颈癌细胞的能力通过铬释放测定来测试。将10x6个靶细胞(负载有肽的HLA匹配的B细胞，不含肽的B细胞)与51Cr(100μCi)孵育1小时，然后与来自在ELISpot测定中针对负载有肽 p16INK4a_37-63的靶细胞反应的一名代表性宫颈癌患者的不同比率的T细胞孵育1小时。被T细胞特异性裂解的靶细胞可通过检测释放的放射性来测量。
可示出在ELISpot测定中针对负载有肽p16INK4a_37-63的靶细胞反应的宫颈癌患者的CD8+T细胞能够裂解HLA-匹配的并且负载有p16INK4a_37-63-肽的B细胞以及宫颈癌细胞系HeLa和Caski(均为HPV和p16INK4a阳性)，而具有相同的HLA匹配而不含p16INK4a_37-63肽的B细胞以及其它HPV-和p16INK4a阴性细胞系未检测到裂解(图3)。这些结果证明p16INK4a-肽特异性T细胞的细胞毒性活性并且证明p16INK4a-表位呈递在宫颈癌细胞上并且被患者的p16INK4a反应性T细胞克隆识别。
实施例4
针对p16INK4a的体液免疫响应
超过900份血清的分析进一步证明一部分个体形成特异性结合于p16INK4a衍生表位的抗体(图4)(Reuschenbach等人，2008)，这示出p16INK4a能够诱导体液免疫响应。
进一步证明了肽p16INK4a_37-63似乎也诱导体外T细胞的最强的干扰素γ分泌，抗体可在血清中检测到。总计374份来自患有宫颈癌、小细胞肺癌、头颈癌的患者以及健康个体的血清在肽ELISA中进行了测试。在4℃下，将肽(20 ug/ml)涂布在96孔微量滴定板(Maxisorp，Nunc，罗斯基勒，丹麦)中过夜，非特异性结合位点用0.5％的酪蛋白封闭并以1:100的抗体：肽测试血清。结合的血清抗体通过HRP-缀合的抗人IgG抗体(Jackson Immuno，West Grove，USA) 和TMB基质(Sigma Aldrich，St.Louis，USA)检测。在标准化光密度为0.03 的背景的截断值处，15％(56/374)的测试血清具有针对p16INK4a_37-63的抗体。
综上所述，这些观察证明患有HR-HPV诱导的发育不良或瘤形成的患者能够形成抗-pl6INK4a-免疫响应。如果该免疫响应将能够消除HR-HPV转化的细胞，则p16INK4a抗原可代表预防和/或治疗表达p16INK4a的瘤形成(前)，特别是 HR-HPV诱导的肿瘤的即将到来的疫苗的非常有吸引力的候选者。
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Claims (20)
1.一种细胞周期蛋白依赖性激酶抑制剂p16INK4a的片段用于生产预防或治疗表达p16的瘤形成前、瘤形成或肿瘤的疫苗的用途，所述片段能够诱导针对p16INK4a的免疫响应且由LPNAPNSYGRRPIQVMMMGSARVAELL组成，其中所述免疫响应包括针对p16INK4a的特异性T细胞响应。
2.根据权利要求1所述的用途，其中所述免疫响应包括CD8-阳性T细胞、CD4-阳性T细胞和抗体的诱导。
3.根据权利要求1所述的用途，其中所述表达p16的肿瘤为晚期癌。
4.根据权利要求1所述的用途，其中所述表达p16的肿瘤为晚期肉瘤。
5.根据权利要求1所述的用途，其中所述肿瘤为阴肛部肿瘤或头颈肿瘤。
6.根据权利要求1所述的用途，其中所述肿瘤为食管-胃-肠肿瘤或肝-胰-胆道肿瘤、皮肤肿瘤或呼吸道肿瘤。
7.根据权利要求1所述的用途，其中所述肿瘤为肾和泌尿道肿瘤、乳腺肿瘤、卵巢肿瘤、子宫内膜肿瘤或前列腺肿瘤。
8.根据权利要求1所述的用途，其中所述肿瘤为神经系统肿瘤，所述神经系统肿瘤包括脑肿瘤。
9.根据权利要求1所述的用途，其中所述肿瘤为人乳头瘤病毒HPV诱导的肿瘤。
10.根据权利要求5所述的用途，其中所述阴肛部癌为宫颈癌。
11.根据权利要求1所述的用途，其中所述疫苗还包括免疫佐剂。
12.一种编码LPNAPNSYGRRPIQVMMMGSARVAELL的核酸序列或含有所述核酸序列的载体用于生产预防或治疗表达p16的瘤形成前、瘤形成或肿瘤的疫苗的用途，所述疫苗能够诱导针对p16INK4a的免疫响应，其中所述免疫响应包括针对p16INK4a的特异性T细胞响应。
13.根据权利要求12所述的用途，其中所述载体为质粒或病毒载体。
14.根据权利要求13所述的用途，其中所述病毒载体为痘病毒、腺病毒、逆转录病毒、疱疹病毒、基于甲病毒属的载体或腺相关病毒(AAV)。
15.根据权利要求14所述的用途，其中所述痘病毒为痘苗病毒或禽痘病毒。
16.根据权利要求14所述的用途，其中所述痘病毒为NYVAC。
17.根据权利要求14所述的用途，其中所述痘病毒为鸟痘病毒。
18.根据权利要求14所述的用途，其中所述痘病毒为金丝雀痘病毒。
19.根据权利要求14所述的用途，其中所述痘病毒为ALVAC。
20.根据权利要求14所述的用途，其中所述痘病毒为TROVAC。
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/EP2012/005155 WO2014090266A1 (en) | 2012-12-13 | 2012-12-13 | P16ink4a derived peptides for prophylaxis and therapy of hpv-associated tumors and other p16 expressing tumors |
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| KR (1) | KR101665410B1 (zh) |
| CN (1) | CN104837497B (zh) |
| BR (1) | BR112015013863A2 (zh) |
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| WO (1) | WO2014090266A1 (zh) |
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| WO2016026919A1 (en) * | 2014-08-20 | 2016-02-25 | Ruprecht-Karls-Universität Heidelberg | Vaccines and manufacturing methods for treatment and prevention of disease |
| CN106046156A (zh) * | 2016-06-02 | 2016-10-26 | 吉林大学 | 肿瘤相关基因cdkn2a表位多肽单克隆抗体制备及其应用 |
| CN108558996A (zh) * | 2018-01-11 | 2018-09-21 | 广州江元医疗科技有限公司 | 一种与p16蛋白特异性结合的单克隆抗体及其在试剂盒中的应用 |
| CN113321720B (zh) * | 2021-03-24 | 2022-03-01 | 深圳市新靶向生物科技有限公司 | 一种与肝癌驱动基因突变相关的抗原肽组合及其应用 |
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| DK2573105T3 (en) * | 2011-09-21 | 2015-12-21 | Ruprecht Karls Universität Heidelberg | p16INK4a-derived peptides for the prevention and therapy of HPV-associated tumors and other tumors expressing p16INK4a |
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- 2012-12-13 WO PCT/EP2012/005155 patent/WO2014090266A1/en active Application Filing
Non-Patent Citations (3)
| Title |
|---|
| Characterization of humoral immune responses against p16, p53, HPV16 E6 and HPV16 E7 in patients with HPV-associated cancers;Miriam Reuschenbach 等;《Int. J. Cancer》;20080910;第123卷;摘要，图2，第2627页右栏第2段至第2630页左栏第1段 * |
| Immune Therapy of HPV-induced Cancers;Oryx GmbH & Co. KG;《https://clinicaltrials.gov/ct2/show/study/NCT01462838》;20111025;全文 * |
| P16INK4a is a tumor autoantigen in HR-HPV induced cancers;Nicolas A. Wentzensen等;《Cancer Res》;20050501;第65卷;1214 * |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2015115137A (ru) | 2017-01-18 |
| WO2014090266A1 (en) | 2014-06-19 |
| RU2626542C2 (ru) | 2017-07-28 |
| KR20150084059A (ko) | 2015-07-21 |
| KR101665410B1 (ko) | 2016-10-12 |
| BR112015013863A2 (pt) | 2017-07-11 |
| CN104837497A (zh) | 2015-08-12 |
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