CN105247365B - 用于癌症治疗的预测性生物标记物 - Google Patents
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- CN105247365B CN105247365B CN201480024805.8A CN201480024805A CN105247365B CN 105247365 B CN105247365 B CN 105247365B CN 201480024805 A CN201480024805 A CN 201480024805A CN 105247365 B CN105247365 B CN 105247365B
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Abstract
本发明大体上涉及根据癌症患者是否将对特异性治疗有反应来识别癌症患者。更具体地,本发明涉及一种用于识别对TLR‑9激动剂治疗的反应者的方法和工具。
Description
说明书
技术领域
本发明大体上涉及根据癌症患者是否将对特异性治疗有反应而对癌症患者进行识别。更具体地,本发明涉及一种用于识别对包含免疫活化DNA构建体的治疗的反应者的方法和DNA构建体,以及待用于治疗癌症患者的DNA构建体,所述患者在采用所述构建体进行治疗开始之前单独地表征为可能的反应者。
背景技术
生物标记物是在血液或其他体液、组织中发现的物质,或作为细胞上的反应者,其预示症状或疾病例如类似癌症的存在与否。生物标记物可以分为预测、预后和药效生物标记物。
预测标记物被用于评估患者会从特定的治疗中反应或收益的可能性。预后标记物允许对肿瘤进行分类,考虑它们的攻击潜力以指导对其治疗或决定如何积极地治疗。药效生物标记物检测药物对肿瘤的短期治疗效果,并且可以用于在新的抗癌药物的临床开发阶段指导剂量选择。
靶向治疗是作用于分子水平的阻止癌症生长或扩散的治疗方法。为了避免不必要的途径,因为这样的治疗通常涉及使用非常有攻击性的药物,需要能够预测患者会不会对靶向治疗有反应的工具。
鉴于这样的事实,即治疗变得越来越具有靶向特异性,生物标记物的地位将会提升。它们将帮助实现个体化治疗性途径和打开个体化肿瘤学的道路。甚至从非反应者中区分对特异性治疗的反应者的可能性将帮助避免患者经受不必要的治疗。
一种广泛已知且完全批准的癌症治疗方法为化疗法。化疗法的主要缺陷是非常具有攻击性药物的使用引起了严重的副作用。为了将副作用最小化,在将化学治疗剂的剂量最小化方面进行了许多努力,例如将化学治疗剂与免疫活化剂联合。一种方法是使用免疫活化DNA。
所谓的未甲基化CG序列已经显示对活化免疫系统非常有效(Krieg AM,Yi AK,Matson S,Waldschmidt TJ,Bishop GA,Teasdale R,Koretzky GA,Klinman DM;CpGmotifs in bacterial DNA trigger direct B-cell activation;Nature 1995 Apr 6374:6522 546-9)。那些序列是从细菌中提取的。EP 1 196 178公开了一种共价闭合环DNA带有部分自补序列,得到了具有带有两端包含未甲基化CG基序的单链环的双链茎的DNA构建体。
EP 1 776 124中提出了使用EP1 196 178的哑铃形DNA构建体与化学治疗剂联合以治疗癌症。被EP 1 196 178的DNA构建体治疗的患者随后接受化学治疗剂。结果是通过所公开的联合化学治疗剂的用量被减少。但是该文件完全没有公开关于识别患者会不会对使用的联合治疗有反应的工具。
本发明的目的是提供一种预测是否癌症患者会对免疫活化DNA的治疗有反应的方法。本发明的另一目的是免疫活化DNA本身,其可以用于这样的方法,即在治疗开始之前治疗被识别为反应者的患者。
发明概述
本发明提供了一种通过确定活化的自然杀伤T(NKT)细胞的频率来预测或监测患癌症或自身免疫疾病的患者是否对TLR-9激动剂的治疗有反应的方法。优选确定用于活化的NKT细胞的免疫分型的分化群集或指定群集(CD)分子CD3+/CD56+/CD69+。整个NKT细胞群体的活化的NKT细胞的比率还可以被确定以评估是否患者会对TLR-9激动剂的治疗有反应的可能性。
其意图在于TLR-9激动剂是DNA构建体,其包括至少一个序列基序N1N2CGN3N4,其中N1N2和N3N4是A、C、T和G的任意组合,并且C为脱氧胞苷,G是脱氧鸟苷,A是脱氧腺苷且T是脱氧胸苷。
对TLR-9激动剂治疗有反应者具有的活化的NKT细胞频率为活化的NKT细胞在全部NKT细胞群体中占至少3%。
进一步的意图是可进行先前的使用非DNA药物的诱导治疗。这样的治疗包括使用或不使用血管再生抑制剂的化学治疗或抗体的使用。
本公开的另一目的是TLR-9激动剂在癌症治疗方法中的使用,特征在于患者具有增加水平的活化NKT细胞。
意图是使用的TLR-9激动剂包括至少一种DNA构建体,其包括至少一个序列基序N1N2CGN3N4,其中N1N2和N3N4是A、C、T和G的任意组合,并且C为脱氧胞苷,G是脱氧鸟苷,A是脱氧腺苷且T是脱氧胸苷。所述激动剂进一步特征在于N1N2是选自包含GT、GG、GA、AT或AA的组的元件,N3N4是选自包含CT或TT的组的元件。
所述激动剂可以包括DNA,其为包含单链或双链DNA的线性开链DNA构建体,或是包含至少一个具有单链环的末端的线性双链DNA构建体。代替使用环来保护线性双链DNA的末端,至少一个L-DNA核苷酸可被用作DNA双链的一部分。
序列基序N1N2CGN3N4可以位于所述DNA序列的单链和/或双链区域内。进一步的意图是DNA TLR-9激动剂的至少一个核苷酸被选自以下的官能团修饰,所述官能团选自包括羧基、胺基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基、硫醇基和醛基的组。
包括DNA构建体的激动剂可以连接到化合物,所述化合物选自包括肽、蛋白质、碳水化合物、抗体、脂质、胶束、囊泡、合成分子、聚合物、微弹、金属颗粒、纳米颗粒或固相的组。
进一步的意图是包括DNA构建体的激动剂可为药物组合物的一部分,其中所述药物组合物可以为疫苗。
本公开的另一目的为预测或监测患癌症或自身免疫疾病的患者是否对TLR-9激动剂的治疗有反应的试剂盒,其包括对活化的NKT细胞在全部NKT细胞群体中的频率进行检测和定量的工具(means)。
所述试剂盒的意图在于所述TLR-9激动剂可以包括DNA构建体,所述DNA构建体包括至少一个序列基序N1N2CGN3N4,其中N1N2和N3N4是A、C、T和G的任意组合,并且C为脱氧胞苷,G是脱氧鸟苷,A是脱氧腺苷且T是脱氧胸苷。
附图说明
图1:verum患者的活化NKT细胞的ROC曲线。
图2:活化NKT细胞对敏感度和特异度。
图3:a:对verum和安慰剂的生物标记物阳性患者的Kaplan-Meier图;b:在verum和安慰剂组的生物标记物阴性患者的Kaplan-Meier曲线。
图4:a:verum组的生物标记物阳性患者对生物标记物阴性患者的Kaplan-Meier图;b:安慰剂组的生物标记物阳性患者对生物标记物阴性患者的Kaplan-Meier图。
发明详述
本发明公开的含义中MGN1703表示共价闭合的部分自互补的DNA链的DNA构建体,所述DNA链具有双链茎和带有未甲基化的CG基序的单链端部环。
根据本公开的“茎”应当理解为DNA双链,其通过同一DNA分子内(其然后部分自我互补)或不同DNA分子内(其部分或完全互补)的碱基配对形成。分子内碱基配对指的是在同一分子内的碱基配对,而不同DNA分子之间的碱基配对称作分子间碱基配对。
在本公开的含义中,“环”应当理解为在茎结构内部或其末端的未配对的、单链的区域。“发夹”是茎和环的不同组合,其在同一DNA分子的两个自我互补的区域杂交形成具有未配对的茎时出现。
“哑铃状”描述在茎区域侧翼两端都具有发夹的线性DNA构建体。因此,本公开上下文中的“线性DNA构建体”描述在双链DNA茎两端包含单链环的线性哑铃状DNA构建体。
本公开的免疫调节指的是免疫活化和免疫抑制。免疫活化优选地指的是活化免疫系统的效应细胞以便其增殖、迁移、分化或以任何其它方式变得有活性。例如无共刺激信号时可以通过免疫活化DNA分子诱导B细胞增殖,而所述B细胞增殖通常需要来自辅助T细胞的共刺激信号。
在另一方面,免疫抑制应当理解为减低免疫系统的活化或效力。通常故意诱导免疫抑制以防止例如移植器官的排斥,以治疗骨髓移植后的移植物抗宿主病,或用于治疗自身免疫病,例如类风湿关节炎或克罗恩氏病(Crohn’s disease)。
在这个背景下,免疫调节还可以指对免疫反应本质或特性的影响,其通过影响仍在发育或成熟的免疫反应或通过调节已经建立的免疫反应的特性。
本公开使用的术语“疫苗接种”指的是施用抗原性材料(疫苗)以产生对疾病的免疫。疫苗可以预防或改善许多病原体例如病毒、真菌、原生动物寄生虫、细菌感染的效果,以及预防或改善变应性疾病和哮喘以及肿瘤的效果。疫苗通常含有用于加强免疫应答的一或多种佐剂,例如,免疫活化核酸。通常认为疫苗接种是最有效且经济的预防传染病和其它疾病的方法。
所施用的材料可以,例如是病原体(细菌或病毒)活的但弱化的形式,所述病原体的死亡的或灭活的形式、纯化的材料如蛋白质、编码抗原的核酸、或细胞例如肿瘤细胞或树突细胞。特别是,最近已经开发了DNA疫苗接种。DNA疫苗接种通过将编码抗原的DNA插入人或动物细胞(且表达,引起免疫系统识别)而生效。免疫系统中一些识别表达的蛋白的细胞将发动对这些蛋白和表达这些蛋白的细胞的攻击。DNA疫苗的一个优势是其非常易于生产和储存。此外,相对于常规疫苗,DNA疫苗具有多种优势,包括诱导更宽范围的免疫应答类型的能力。
疫苗接种可以用作预防性方法,在疫苗接种的健康个体暴露于抗原后产生针对所述抗原的免疫力。或者,治疗性疫苗接种通过将经疫苗接种的患病个体的免疫系统导向抗原,可以导致所述免疫系统应答的加强。预防性和治疗性疫苗接种均可以应用于人类以及动物。
术语“癌症”包括被治疗或预防的癌性疾病或肿瘤,其选自包括但不限于乳腺癌、黑色素瘤、皮肤肿瘤、淋巴瘤、白血病、胃肠肿瘤,包括结肠癌(colon carcinomas)、胃癌、胰腺癌、结肠癌(colon cancer)、小肠癌、卵巢癌、宫颈癌、肺癌、前列腺癌、肾细胞癌和/或肝转移的组。
本公开的自身免疫病包括类风湿关节炎、克罗恩氏病、全身性红斑狼疮(SLE)、自体免疫甲状腺炎、自身免疫性甲状腺炎、多发性硬化、格雷夫斯病、重症肌无力、乳糜泻和阿迪森氏病。
在使用单链末端环中具有未甲基化CG序列的免疫活化哑铃状DNA构建体的实验中,得出免疫活化和效应物途径的特定细胞类型的频率升高与使用DNA构建体的成功治疗相关。
在脊椎动物中,所谓的“toll样受体(TLR)”是其天生免疫系统的部分。TLRs是特异性免疫受体的家族,当它们感测到高度保守的病原体相关的分子模式时会引起保护性免疫反应,分子模式例如为蛋白质、脂质结构、碳水化合物结构和某些核酸。对几种TLRs的合成的激动剂,包括TLR-3、TLR-4、TLR-7、TLR-8和TLR-9已经被发展用于癌症的治疗,通常用于肿瘤存在时活化免疫系统。TLR-9识别包含未甲基化CG的DNA序列的存在,其通常在细菌中被发现,但是实际上不在人类染色体组的DNA。因此包含未甲基化CG的DNA序列已经被设计为人造TLR-9激动剂。这样的包括未甲基化CG的DNA构建体的效应取决于它们与TLR-9的相互作用,并且DNA-蛋白质的相互作用取决于DNA和蛋白质两者的构象。实验数据表明哑铃状DNA分子出人意料的适用于诱导免疫应答。
为了识别预测是否癌症患者会对TLR-9激动剂的施加有反应的工具,使用了在单链末端环中具有未甲基化CG基序的哑铃状DNA构建体。
总计46位患有转移性结直肠癌的患者被选择进行研究,他们先前已经采用标准一线联合治疗方法(使用或没有使用人体单克隆抗体抑制血管内皮细胞生长因子A)治疗了4.5-6个月。在约1-6星期的无治疗间隔后,对患者进行了随机选择,这样32个患者接受了每剂量60mg的DNA构建体MGN1703,并且14个患者接受了安慰剂,每个患者每周两次皮下注射。
在12星期的治疗后,所有的患者对肿瘤发展进行了检查。基于肿瘤发展的存在或缺少,患者被分为两组,称为“PFS组”。肿瘤没有发展的患者被称为未发展患者且标记为“PFS1”,同时肿瘤发展了的患者标记为“PFS0”。显然,肿瘤没有发展(PFS1)显示了对治疗有反应的可能性,同时肿瘤生长(PFS2)显示了缺少反应。对每个患者继续治疗直到发现肿瘤的发展。
表1总结了结果。显然几乎所有的没有发展的患者都接受了DNA构建体。
表1:患者的名称
在MGN1703或安慰剂的第一次施用之前(位于基线),收集患者的血液样品。下面的免疫变量的分布,例如整个PBMC群体的部分以及活化的和未活化的子群体的关系被确定。调查了12星期后的各PFS组名称的相关性和基线处的所有免疫变量。确定了下列免疫变量:单核细胞、活化的单核细胞1、活化的单核细胞2、B细胞、活化的B细胞,T细胞,活化的T细胞,天然杀伤(NK)细胞、活化的NK细胞、NKT细胞、活化的NKT细胞、浆细胞样树突细胞(pDCs)、活化的pDCs、骨髓状树突细胞(mDCs)、和活化的mDCs。表2总结了细胞类型、CDs和确定的频率。
表2:细胞类型、CDs和确定的频率的关系
为了评估是否免疫变量中的一个可以用作正确的生物标记物,所谓的Cox回归被用于计算每个免疫变量。Cox回归允许不考虑任何冒险函数的情况下估计参数的效果。从这样的计算中得到的冒险率可以为低于1,并且涉及显著的p值低于0.05。另一方面,观察到的效果不涉及施加的TLR-9激动剂。那些标准仅施加到活化的NKT细胞,具有冒险率为大约0.933和p值为0.0309。
出人意料的,活化的NKT细胞的百分比可以被用于预测verum组的治疗成功结果。活化的NKT细胞的百分比和PFS组状况之间的关系采用先进的、已建立的统计学分析在verum组中进行研究。
受试者工作特性(ROC)曲线、或单纯的ROC曲线,显示了二进制分类系统的性能,同时它的辨别阈值是变化的。ROC曲线通过绘制在不同阈值设置下从阳性中得出的真阳性的分数对从阴性中得出的假阳性而得到。真阳性也称为灵敏度,并且假阳性为1减去特异度或真阴性率。
几十年来ROC分析被用于药物、放射学、生物测定学、和其他领域,并且正越来越多地用于机器学习和数据挖掘的研究。在生物标记物中,它可以用于研究是否潜在的生物标记物可以具有临床效力,也就是说它可以用于预测目的。成功的诊断实验或生物标记物将得到曲线,其弯曲在对角线上方,同时不成功的实验将反射对角线,或落到对角线下面。因此,ROC曲线提供了诊断实验是否成功的信息。
verum患者的活化的NKT细胞的ROC曲线被建立(图1)。曲线下面的区域被确定为0.71,其为生物标记物的可靠度的清楚的指示。约登指数,其可以用于确定实验读数的最佳界限值,显示了最佳界限为3.08%活化的NKT细胞。图2显示了活化的NKT细胞对灵敏度和特异度。
所有的患者现在根据他们的活化NKT细胞的水平被分类成组。细胞水平高于界限3.08%活化的NKT细胞的患者被称为生物标记物阳性,同时低于界限水平的患者被称为生物标记物阴性。
图3a显示了verum和安慰剂的生物标记物阳性患者的Kaplan-Meier图(实线:用MGN1703治疗的患者;虚线:用安慰剂治疗的患者)。显然生物标记物阳性组的存活可能性出人意料地与TLR-9激动剂的施用有关。
图3b显示了verum和安慰剂的生物标记物阴性患者的Kaplan-Meier图。显然在该verum组内的未发展存活时间明显短于生物标记物阳性verum患者(相比图3a)。
图4a显示了生物标记物阳性患者对生物标记物阴性患者的Kaplan-Meier图,仅从verum治疗的组中得到(实线:生物标记物阳性患者;虚线:生物标记物阴性患者)。明显地,生物标记物阳性患者相比生物标记物阴性患者在存活可能性上有明显优势,甚至是当两组都接受verum治疗MGN1703时。
图4b显示了生物标记物阳性和阴性患者的安慰剂组的Kaplan-Meier图(实线:生物标记物阳性患者;虚线:生物标记物阴性患者)。明显地当两组均仅用安慰剂治疗时,两组均显示出或多或少相同的未发展存活。明显地,生物标记物适合于评估患者是否是TLR-9激动剂的治疗的反应者。另外,安慰剂组显示了生物标记物与患者的全部健康状况引起的效应无关。
本发明提供了一种新型的预测性生物标记物,其针对采用TLR-9激动剂,特别是具有带未甲基化CG基序的单链末端环的双链茎的共价闭合的自补DNA链治疗癌症的反应者。基线处的活化的NKT细胞(CD3+/CD56+/CD69+)的频率的确定允许评估是否患者是对DNA构建体的治疗的反应者的可能性。重要的是,在安慰剂组,带有反应者类特性的患者,表现了与非反应者相同的方式,显示了生物标记物阳性verum患者的延长的未发展存活时间实际上归因于选择的治疗的生物标记物的施用,不只是更好的全身健康或任何非特异性效应。
材料和方法
样品处理
FACS的全血(10mL)收集在StreckBCT试管中。在血液样品取样后2小时内送至分析实验室。根据建立的实验,样品被存放于室温。评估浆细胞样树突细胞(pDCs)、骨髓状树突细胞(mDCs)、单核细胞、天然杀伤(NK)细胞、NKT细胞、B细胞、T细胞和其他细胞群体的频率和活化状态。
分析方法
荧光活化细胞分类(FACS)根据建立的原理实施。全血样品用荧光标记抗体进行染色并且被孵育。免疫细胞的表型分析采用FACScalibur(Becton Dickinson)流式细胞仪实施。各分析的细胞群体的频率存档为患者的各样品。
对特定细胞群体的活化的人PBMC的分析
浆细胞样树突细胞(pDC)的CD40表达
细胞用下列单克隆抗体的组合进行染色:抗-系列标记-FITC,(包含针对CD3、CD14、CD16、CD19、CD20、CD56的抗体的抗体鸡尾酒);抗-CD123-PE;抗-HLA-DR-PerCP;抗-CD40-APC;PDC被定为:谱系阴性,HLA-DR阳性,CD123阳性细胞。在PDC群体中CD40被用作活化标记物。
使用了活化标记物CD69的NK-、NK-T和T细胞的活化
细胞用下列单克隆抗体的组合来染色:抗CD3-FITC;抗CD56-PE;抗CD69-APC。
NK细胞被定为:CD3阴性、CD56阳性细胞
NK-T细胞被定为:CD3阳性、CD56阳性细胞
T细胞被定为:CD3阳性、CD56阴性
CD69被用作所有3个群体的活化标记物。
骨髓状树突细胞(mDC)的CD86表达:
细胞用下列单克隆抗体的组合进行染色:抗-系列标记-FITC,(包含针对CD3、CD14、CD16、CD19、CD20、CD56的抗体的抗体鸡尾酒);抗-CD11c-PE;抗-HLA-DR-PerCP;抗-CD86-APC
MDC被定为:谱系阴性,HLA-DR阳性,CD11c阳性细胞。
在MDC群体中CD86被用作活化标记物。
B细胞和单核细胞的CD86表达;单核细胞的CD169表达
细胞用下列单克隆抗体的组合进行染色:抗CD14-FITC;抗CD169-PE;抗CD19-PerCP;抗CD86-APC
B细胞被定为CD19阳性细胞。在B细胞群体中CD86被用作活化标记物。
单核细胞被定为CD14阳性细胞。在单核细胞群体中CD86和CD169被用作活化标记物。
Claims (18)
1.用于检测和定量CD3+/CD56+/CD69+的活化的自然杀伤T(NKT)细胞的频率的工具在制备用于预测或监测患癌症的患者是否对TLR-9激动剂的治疗有反应的试剂盒中的用途。
2.根据权利要求1的用途,其中所述TLR-9激动剂为包括至少一个序列基序N1N2CGN3N4的DNA构建体,其中N1N2和N3N4是A、C、T和G的任意组合,并且C为脱氧胞苷,G是脱氧鸟苷,A是脱氧腺苷且T是脱氧胸苷。
3.根据权利要求1或2的用途,其中确定活化的NKT细胞在全部NKT细胞群体中所占的比例。
4.根据权利要求1或2的用途,其中对TLR-9激动剂治疗有反应者具有的活化的NKT细胞频率为活化的NKT细胞在全部NKT细胞群体中占至少3%。
5.根据权利要求1或2的用途,其中预先进行非DNA药物的诱导治疗。
6.根据权利要求1或2的用途,用于确定是否所述患者具有升高的活化的NKT细胞水平。
7.根据权利要求1或2的用途,其中N1N2是选自包含GT、GG、GA、AT或AA的组的元件,N3N4是选自包含CT或TT的组的元件。
8.根据权利要求2的用途,其中所述DNA构建体是包含单链或双链DNA的线性开链DNA构建体,或是包含至少一个具有单链环的末端的线性双链DNA构建体。
9.根据权利要求2的用途,其中序列基序N1N2CGN3N4位于所述DNA构建体的单链和/或双链区域内。
10.根据权利要求1或2的用途,其中所述TLR-9激动剂包括至少一个L-DNA核苷酸。
11.根据权利要求2的用途,其中至少一个核苷酸被官能团修饰,所述官能团选自包括羧基、胺基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基、硫醇基和醛基的组。
12.根据权利要求2的用途,其中所述DNA构建体连接化合物,所述化合物选自包括碳水化合物、脂质、胶束、囊泡、合成分子、聚合物、微弹、或固相的组。
13.根据权利要求12的用途,其中所述聚合物为肽或蛋白质。
14.根据权利要求12的用途,其中所述固相为金属颗粒或纳米颗粒。
15.根据权利要求2的用途,其中所述DNA构建体连接抗体。
16.根据权利要求1或2的用途,其中所述TLR-9激动剂为药物组合物的一部分。
17.根据权利要求16的用途,其中所述药物组合物为疫苗。
18.根据权利要求1的用途,其中所述用于检测和定量CD3+/CD56+/CD69+的活化的自然杀伤T细胞的频率的工具为抗CD3-FITC、抗CD56-PE和抗CD69-APC。
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