CN1346284A - 用于治疗肿瘤的cd40结合分子和ctl肽 - Google Patents
用于治疗肿瘤的cd40结合分子和ctl肽 Download PDFInfo
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- CN1346284A CN1346284A CN99807669A CN99807669A CN1346284A CN 1346284 A CN1346284 A CN 1346284A CN 99807669 A CN99807669 A CN 99807669A CN 99807669 A CN99807669 A CN 99807669A CN 1346284 A CN1346284 A CN 1346284A
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Abstract
本发明公开了一种治疗肿瘤或传染病的方法和组合物,其中该组合物包括CD40结合分子以及CTL活化肽,例如,肿瘤抗原。该组合物用于增强肽肿瘤疫苗的抗肿瘤效力,或者用于活化CTL以便活化的CTL可起抗肿瘤细胞或感染细胞的作用。CD40结合分子包括抗体分子,以及其同源物,类似物和修饰的或衍生的形式,包括如Fab,(Fab’)2和Fv的免疫球蛋白片断,以及包括结合CD40并活化CTL反应的肽,寡核苷酸,肽模拟物和有机化合物的其它分子。
Description
发明领域
本发明涉及药物组合物中的CD40结合分子和激活CTL的肽，包括肿瘤抗原。
发明背景
许多肿瘤逃避了我们免疫系统的检测。在癌症患者中在缺失肿瘤细胞的免疫系统特异性机理中清楚地存在定量和/或定性缺陷。通过能够识别和杀伤被病毒感染或转变成癌细胞的细胞的细胞毒性T细胞(CTL)提供了这些机理之一。以前假定树突状细胞(DC)刺激T-辅助细胞，该辅助细胞依次对CTL的活化提供帮助。本发明人已证实，T-辅助细胞不直接对该CTL提供辅助信号(通过分泌IL2)，然而，该T-辅助细胞对诱导在没有T-辅助细胞的情况下能够激活CTL的还未鉴定的细胞表面和/或可溶性分子的DC提供一信号。通过该T-辅助细胞提供给该DC的该信号通过CD40L-CD40相互作用介导。这种新发现对癌症免疫疗法提供了一种极难得的机会。
当自身细胞受到病毒感染时或者当它们转变成癌细胞时，免疫系统能够杀死这些自身细胞。这种潜在危险的机理必需清楚地在严格控制之下。该免疫系统的CTL以无活性前体循环。为了将其激活，该前体T-杀伤细胞必需识别其特异的抗原肽，该抗原肽以MHC I型分子存在于专职APC上。然而，为了引发这些T细胞，该APC还必需以尤其通过共有刺激的表面分子B7.1和B7.2以及通过淋巴因子IL-12提供的固有共同刺激的前后序列呈递该抗原。
直到最近，据信需要一在相同APC上识别相同抗原的T-辅助细胞完全激活该CTL。该特异T-辅助细胞应供应例如活化该CTL所需的IL-2的细胞因子。然而，Guerder和Matzinger(J.Exp.Med.176：553(1992))提出了用于CTL活化的“特许”模型。在该模型中，暗示当T-辅助细胞在专职APC上识别其抗原时，该T-辅助细胞将对该APC传递一活化信号，结果随后能够在不需要T-辅助细胞存在的情况下激活CTL。就在不久前，已对该特许模型的分子机理进行了解释。Schoenberger等人(Nature 393：480(1998))描述了该CD40L-CD40途径在该特许模型中的作用。T-辅助细胞和DC之间通过该CD40-CD40L结合的相互作用致使该DC活化，因此使该DC能够有效地引发原初CTL。
DC通过我们身体的组织循环并以这种方式能够收集、加工和呈递抗原。收集抗原之后，它们移动到该引流淋巴结，在其中它们向该T细胞呈递抗原。众所周知，DC需要最适地被激活。静息DC仅表达普通级MHC和共同刺激的分子并且为T细胞的差的刺激物。DC能够被炎症细胞因子和细菌产物激活，这将导致MHC和共同刺激的分子正调节。DC活化成完全成熟的DC、表达最佳级MHC分子、共同刺激的分子和如IL-12的淋巴因子，这需要通过该CD40受体附加地触发这些细胞。因此，在吸收抗原位置处的炎症细胞因子和T-辅助细胞相互作用过程中的CD40L-CD40相互作用的组合致使特许该DC活化CTL的能力达到最佳。
许多肿瘤逃避特异CTL机理的免疫检测。如果在没有炎症的情况下DC富集肿瘤抗原，那么被激活的T-辅助细胞的数量可能降低至足够诱导待激活的CTL，从而诱导一适宜的抗肿瘤反应。这一观念已提示研究人员通过给药直接刺激CTL活性的例如IL-2和IL-12的细胞因子或通过给药用GM-CSF转染的肿瘤细胞加强抗原呈递来帮助该免疫系统。这些策略已满足各种仅启动的结果。
很显然，仍有很大需求寻找产生和/或提高涉及细胞和体液免疫的保护性抗肿瘤反应。发现该CD40活化途径是一种产生一级体液和细胞免疫反应的主要免疫调节途径。如上所述，在DC上的该CD40途径担负着诱导抗肿瘤CTL反应。此外，该CD40途径在巨噬细胞上的活化刺激一强的杀肿瘤活性。
发明概述
本发明包括药物组合物中的CD40结合分子和激活CTL的肽，包括肿瘤抗原。将这种组合物用于提高肽肿瘤疫苗的抗肿瘤效应，或者用于另外激活CTL，以便该激活的CTL能够起到抵抗肿瘤细胞或受感染的细胞的作用。该CD40结合分子可以包括抗体分子，及其同系物、类似物和其修饰或衍生形式，包括象Fab、(Fab′)2和Fv的免疫球蛋白片段，以及结合在CD40上并激活该CTL反应的包括肽、低聚核苷酸、拟态肽和有机化合物的小分子。激活CTL的肽包括来自腺病毒且具有序列SGPSNTPPEI(SEQ ID NO：2)的E1A-肽和来自人乳头瘤病毒16型且具有序列RAHYNIVTF(SEQ ID NO：3)的HPV16 E7-肽。
可以通过包括注射的适宜方式将该CD40结合分子和激活CTL的肽给药到一患者，或者可以给药编码这种分子和肽的基因构建体，并且因此在体内或体外生产该分子和肽。根据本领域中众所周知的方法进行这种基因疗法。如果在体外进行该基因构建体的转染或感染的话，可以向该患者给药该被感染或转染的细胞，或者可以在体内进行这些步骤，由此内生地生产该分子和肽。如果在体外进行，可以通过包括电穿孔、磷酸钙转染、微注射的传统方法或者通过将该基因构建体加入适宜脂质体中进行该转染或感染。可以使用包括反转录病毒、腺病毒或细小病毒的载体或质粒加入该基因构建体，然后它们在体内或体外表达。
本文证实，通过CD40-CD40配体(CD40L)相互作用介导对CTL引发有帮助的T-细胞，在没有CD4+T细胞的情况下CTL引发的缺少可以通过在有包括肿瘤抗原的激活CTL的肽的情况下来自骨髓的APC的单克隆抗体(mAb)所介导的CD40活化来恢复。而且，由CD4+T细胞表达的CD40L的阻断导致不能产生CTL免疫。这种缺陷可以通过体内CD40-触发来克服。CD40的体内触发可以明显地提高肽基抗肿瘤疫苗的功效，或者另外激活CTL，从而导致抗肿瘤或抗感染的细胞反应。
还应指出的是，激活CTL的肽能够成为耐受原的，意思是在没有抗-CD40的情况下抵抗表达这种肽的细胞的寄主反应受到抑制。然而，这种肽与激活抗-CD40的抗体组合将耐受性转变成强的CTL活性。而且，如上所述，CD40连接反应能够赋予已经保护的肿瘤-特异性肽疫苗以在载有肿瘤的小鼠中诱导治疗CTL免疫的能力。
这些发现一起证明了，CD40-CD40配体对起一决定是否引发或耐受原初周围CTL的开关的作用，因此可以有效地将例如单克隆抗体和其它刺激配体的CD40结合剂用于与激活CTL的肽组合。
附图简述
图1：E1B特异性CTL的交叉引发需要CD4+T辅助细胞
在不同效应物/靶比值下检测用Ad5E1-BALB/c MEC免疫的正常(a)或缺失CD4的B6(b)的小鼠的脾细胞的载有EIB192-200肽(实线)或Dd受限制的对照肽HPV-16 E749-57(虚线)的同源MEC靶细胞的溶胞作用，该E1B192-200肽来自人体腺病毒并具有序列VNIRNCCYI(SEQID NO：1)。每条线代表一只小鼠。所示数据代表了具有相同结果的三个试验之一。
图2：CD40激活替换CD4+T辅助细胞
将缺失CD4的(a，b)或II型有缺陷的I-Ab敲除(KO)的(c，d)B6小鼠的脾细胞用Ad5E1-BALB/c MEC免疫并用激活CD40的抗体(Ab)FGK45(a，c)或同种型对照抗体(b，d)处理。检测这些脾细胞对载有E1B192-200肽(实线)或HPV-16E749-57对照肽(虚线)的同源MEC靶细胞的E1B特异性CTL活性。每条线代表一只小鼠。所示数据来自两个独立的实验。E/T比为效应物/靶比值。
图3：B细胞对交叉引发APC或交叉引发的抗-CD40介导的恢复不是必需的
从未经处理的(a)、缺失CD4且B细胞有缺陷的B6 MT小鼠(b，c)中取出脾细胞，这些小鼠是用Ad5E1-BALB/c MEC免疫并接受过同种型对照抗体(b)或激活CD40的抗体FGK45(c)。检测这些脾细胞对载有E1B192-200肽(实线)或HPV E749-57对照肽(虚线)的同源MEC靶细胞的E1B特异性CTL活性。每条线代表一只小鼠。所示数据代表两个具有相同结果的两个实验之一。
图4：CD40L阻断阻止E1B特异性CTL的交叉引发
从用Ad5E1-BALB/c MEC免疫并用阻断CD40L的抗体MR-1(a)或对照抗体(b)处理的B6小鼠、或从免疫24小时后用阻断CD40L的抗体MR-1与激活CD40的抗体FGK45一起处理的小鼠(c)中取出脾细胞。检测这些脾细胞对载有E1B192-200肽(实线)或HPV E749-57对照肽(虚线)的同源MEC靶细胞的E1B特异性CTL活性。每条线代表一只小鼠。所示数据代表两个具有相同结果的三个实验之一。E/T比为效应物/靶比值。
图5：皮下注射有E1A-肽的小鼠不再能封固E1A-特异性CTL
用IFA中的20μg E1A-肽(a，b)或对照肽(c，d)给C57BL/6小鼠皮下注射2次(间隔1周)，1天后用SAMB7腹膜内注射刺激(b，d)，该SAMB7为一表达大量E1A-肽的细胞系。检测来自这些小鼠的大块培养物的对用E1A-肽(-■-)或HPV16 E7-肽(-O-)脉冲的靶细胞的E1A-特异性细胞毒性。显示了在不同效应物∶靶(E/T)比值下典型大块培养物的溶胞率。将该实验重复4次，从而获得可比较的结果。
图6：在以IFA皮下注射之后快速地在全身分布耐受的E1A-肽
将从未经处理的C57BL/6小鼠(-)或从16个小时以前经皮下注射了IFA中的100μg E1A-肽或HPV16 E7-肽的小鼠获得的脾细胞用作用于E1A-特异性CTL克隆的刺激物细胞。显示了在没有扣除本底计数的情况下不同效应物：刺激物浓度的[3H]-胸腺嘧啶掺入(cpm)+/-S.E.M。结果为单独试验的代表。
图7：CD40在体内触发之后将CTL诱导耐受回复成CTL引发
对野生型C57BL/6小鼠(a，b)和H-2b CD40-/-小鼠(c，d)仅经皮下注射IFA中的20μg的E1A-肽、与大鼠IgG2a对照抗体一起注射(a)、或者与抗-CD40mAb FGK-45一起注射(b，d)。检测来自这些小鼠的大块培养物对用E1A-肽(-■-)、HPV16 E7-肽(-O-)或转化肿瘤细胞的Ad5EI(-◆-)脉冲的靶细胞的E1A-特异性细胞毒性。显示了在不同E/T比值下典型大块培养物的溶胞率。分别将该实验重复18次(B6小鼠)和2次(CD40-/-小鼠)，从而获得可比较的结果。在(e)中显示了在E/T为60时从仅注射了IFA中的E1A-肽(左边)或注射了IFA中的E1A-肽和抗-CD40mAb(右边)的B6小鼠中分别获得的23块、37块CTL培养物的溶胞率％。显示了每组的平均正标准偏差(E1A相对E1A+抗-CD40：p＜0.01，学生t测验)。
图8：通过肽与体内CD40触发一起的联合治疗的HPV16和E7转化细胞的疗法
对小鼠经皮下注射25.000 HPV16转化同源细胞(TC-1)。将C57BL/6小鼠保持未被处理(-O-)或在6天后以IFA腹膜内注射接受100μg HPV16 E7-肽(-□-)、以IFA腹膜内注射接受100μg HPV16 E7-肽和抗-CD40 mAb FGK-45(-■-)或以IFA腹膜内注射接受对照肽和抗-CD40 mAb FGK-45(-●-)。在(a)中描述了不同处理组(n＝10)中产生肿瘤的小鼠的百分比。经统计用HPV16-肽和抗-CD40 mAb处理的组与其它三个组之间的差异很大(p＜0.01)(Log-Rank试验)。在(b)中显示了存活动物的百分比(E7-肽处理组：E7-肽和抗-CD40处理组：p＜0.002，Log-Rank试验)。
可以通过传统生产和筛选技术制备本发明的CD40结合分子。在Nature 393：474-77(1998)中分别描述了大鼠和仓鼠抗鼠CD40单克隆抗体(“Mab”)并可商购获得(Pharmingen，Inc.，CA)。Rolink.A等人在Immunity 5,319-330(1996)中描述了该抗鼠CD40 MAb，叫做FGK45，将其用于下述试验。可以通过本领域中众所周知的技术制备抗人CD40 MAb，并已由G.Khler和C.Milstein述及(Nature，1975：256：495-497)。可以通过用原初CD40免疫啮齿动物(例如小鼠、大鼠、仓鼠和豚鼠)产生MAb，该原初CD40是在细胞上表达的或是从人血浆或尿中纯化的，或者在真核或原核系统中表达的重组CD40或其片段。可以将其它动物用于免疫，例如非人灵长类、表达人免疫球蛋白的转基因小鼠和用人B淋巴细胞移植的重度联合免疫缺陷(SCID)小鼠。如G.Khler和C.Milstein Id所述，通过融合来自用骨髓瘤细胞(例如Sp2/0和NS0)免疫过的动物的B淋巴细胞的传统方法可以产生杂交瘤。此外，通过从噬菌体展示系统中的人B淋巴细胞中筛选重组单链Fv或Fab库，能够产生抗-CD40 MAb。该MAb对CD40的特异性可以通过酶联免疫吸附测定(ELISA)、蛋白质印迹法或其它免疫化学技术检测。抗体在CTL上的激活活性以及激活CTL的肽可以使用下面实施例中所述的测定来评价。
为了处理人体，应优选将该抗-CD40 MAb以嵌合的、去免疫的、人化的或人抗体使用。这些抗体可以降低免疫原性，因此避免了人抗鼠抗体(HAMA)反应。优选该抗体为IgG4、IgG2或经过其它一般诱变且不增加抗体依赖型细胞的细胞毒性(S.M.Canfield和S.L.Morrison，J.Exp.Med.，1991：173：1483-1491)并且不补充所介导的细胞溶解(Y Xu等人，J.Biol.Chem.，1994：269：3468-3474；V.L.Pulito等人，J.Immunol.，1996；156：2840-2850)的IgG或IgM。
嵌合抗体是通过本领域中众所周知的重组法生产的，并具有动物可变区和人恒定区。人化抗体比嵌合抗体具有更大程度的人肽序列。在人化抗体中，仅对抗原结合和特异性有反应的互补性决定区是来自动物且具有与该动物抗体相应的氨基酸序列，并且该分子中剩余部分基本上全部(在某些情况下除可变区中小部分的构架区之外)都是来自人体且相应于人抗体的氨基酸序列。参见L.Riechmann等人的Nature，1988；332：323-327；G.Winter的US5225539；C.Queen等人的US5530101。
去免疫的抗体为已剔除T和B细胞表位的抗体，如国际专利申请PCT/GB98/01473中所述。当将它们用于体内时它们降低了免疫原性。
可以通过几种不同方式制备人抗体，包括使用人免疫球蛋白表达库(Stratagene Corp.，La Jolla，California)以生产人抗体片段(VH、VL、Fv、Fab或(Fab′)2，以及使用这些片段采用与生产嵌合抗体相似的技术构建整个人抗体。还可以在具有人免疫球蛋白基因组的转基因小鼠中生产人抗体。这些小鼠可以从Abgenix，Inc.，Fremont，California和Medarex，Inc.，Annandale，New Jersey获得。
人们还可以创建连接重链和轻链Fv区的单肽链结合分子。在US4946778中描述了单链抗体(“ScFv”)和其构建方法。或者，可以通过相似方式构建和表达Fab(M.J.Evans等人的J.Immunol.Meth.，1995；184：123-138)。全部和部分的人抗体所有的免疫原性都比不上整个鼠MAb的，并且这些片段和单链抗体也几乎没有免疫原性。因此所有这些类型的抗体都不可能引起免疫或变态反应。因此，它们比整个动物抗体更适宜在人体内给药，特别是当必需重复或长期给药时。此外，更小尺寸的抗体片段可以帮助改善组织生物利用率，而该生物利用率可能对急性病适应症如肿瘤治疗中更好地药量积聚是至关重要的。
基于抗-CD40 mAb或已知激活CTL的肽的可变区的分子结构，人们能够使用分子模型和合理的分子设计产生和筛选分别模拟所述抗体或肽的结合区的分子结构且激活CTL的分子。这些小分子可以是肽、拟态肽、低聚核苷酸或其它有机化合物。可以使用这些模拟分子治疗癌症和感染。另外，人们可以使用本领域中常用的大规模筛选步骤从化合物库中分离适宜分子。
可以从下述的体内试验和测定、或者从动物实验或从人体临床试验通过外推法容易地确定本发明分子的剂量。在为抗体或蛋白质时，尽管某些小分子可能适宜口服，但是本发明的分子优选通过注射给药。下面描述证明本发明效果的测定和试验。
实施例1：在引发CTL 时通过CD40发出的信号能够代替CD4+辅助T细胞
使用一完全鉴定的模型系统探测T-细胞帮助在体内一级活化CD8+CTL反应的机理。用表达人腺病毒5型早期区域1(Ad5EI-BALB/c MEC)的异源BALB/c(H-2d)鼠胚细胞(MEC)免疫的C57BL/6(具有主要组织相容性复合体(MHC)H-2b)小鼠产生强烈的抗腺病毒EIB蛋白(E1B192-200)的H-2Db限制性表位的CTL反应(图1a)。由于通过Ad5EI-BALB/c MEC表达的异源H-2d MHC分子不能引发H-2b限制性宿主CTL，因此必需交叉引发才能产生E1B特异性CTL，即，通过宿主APC吸收并H-2b限制性再呈递抗原。由于缺失CD4+T-辅助(Th)细胞的小鼠在免疫之前不再封固E1B特异性CTL反应，因此EIB特异性CTL的交叉引发为严格辅助依赖型(图1b)。
为了研究通过CD40发出的信号是否能够替代CTL引发中的CD4+辅助T细胞，在用Ad5EIBALB/c MEC免疫之前在体内使小鼠缺失CD4+T细胞。免疫一天后，这些小鼠接受活化抗体抗鼠CD40mAb FGK45或同种型配对的对照抗体的单一注射。向缺失CD4的给药FGK45，经过免疫的小鼠因此有效地恢复了E1B特异性CTL反应(图2a)，然而用对照抗体的处理没有(图2b)。在单用FGK45处理的原初小鼠中E1B特异性CTL的引发没有缺失(未显示)。为了解释FGK45的效果是通过用抗-CD4抗体处理没有缺失的剩余D4+细胞介导的可能性，将缺少成熟功能性CD4+周围T细胞的B6 I-Ab敲除的小鼠用Ad5EI-BALB/c MEC免疫。在这些小鼠中的该免疫反应反射出缺失CD4的小鼠中所看到的，其中仅在接受激活CD40的抗体的小鼠中可以检测出E1B特异性CTL(图2c)，并且在接受对照抗体中不能检出(图2d)。
还研究了在引发CTL时在缺失CD4的小鼠中抗-CD40抗体的需要是否能被细菌性脂多糖(LPS)(50μg静脉内)、促炎细胞因子的有效诱导剂，或通过用Ad5EI-BALB/c MEC免疫之后给药IL-2(在不完全弗氏佐剂中1×105U，皮下)取代。然而用FGK45处理的缺失CD4的小鼠呈现出强的E1B特异性CTL活性，LPS或IL-2处理都不能导致可检出的CTL引发(未显示)。
CD40在B细胞上的连接反应正调节它们的共同刺激活性，这暗示这些细胞在通过用激活CD40的抗体处理恢复引发CTL中的作用。为了解释该问题，将缺少成熟B细胞的B6 MT小鼠用异源Ad5EI-BALB/c MEC免疫。E1B特异性CTL的交叉引发不需要成熟B细胞(图3a)。但是，当缺失CD4+细胞时，该B细胞有缺陷的小鼠不产生E1B特异性CTL反应(图3b)。用FGK45单克隆抗体通过CD40的激化完全恢复了CD4缺失型的能力。MT小鼠引发E1B特异性CTL(图3c)。因此不需要B细胞作为APC或用于这种模型系统中交叉引发的佐细胞，它们也不需要在没有CD4+辅助T细胞的情况下交叉引发CTL时CD40所介导的恢复。这些结果证明了通过CD40的来自骨髓的APC的激活在E1B特异性CTL的交叉引发中可以回避对CD4+T-辅助细胞的需要。
实施例2：阻断CD4+辅助T细胞的能力以通过CD40L-D40径与APC相互作用防止在正常小鼠中的抗原特异性CTL反应
如果该CD40L-CD40相互作用代表帮助CTL的CD4+辅助T细胞所用的生物途径，那么希望阻断CD4+T细胞通过CD40L-CD40相互作用与APC相互作用的能力，从而消除E1B特异性CTL反应在正常小鼠中引发。将B6小鼠用Ad5EI-BALB/c MEC免疫，然后或者用CD40L阻断的抗体MR1或者用对照抗体处理。与接受对照抗体的小鼠中看到的有效CTL引发(图4b)相比，CD40L阻断的结果大大地降低了E1B特异性CTL反应(图4a)。CD40通过FGK45发出信号之后，CD40L阻断所诱导的引发缺陷完全得到恢复(图4c)。因此通过CD40L诱导的CTL引发的缺陷在于TH细胞不能传递(而不是接收)CD40L所介导的信号。
实施例3：在给药肽之后E1B特异性CTL的无应答性
使用前面描述的模型系统(Toes等人，J.Immunol.156：3911(1996))。已说明用IFA中的来自Ad5E1A的CTL表位SGPSNTPPEI(SEQ ID NO：2)皮下接种疫苗防止了小鼠长出表达Ad5E1A的肿瘤。这说明该E1A/IFA疫苗诱导抑制而不是诱导E1A-特异性CTL免疫。而且，向T细胞受体(TCR)转基因的小鼠给药该E1A/IFA，这样表达E1A-特异性CTL克隆的TCRα和β链，因此强烈地抑制了肿瘤-特异性CTL所介导的免疫。这些试验检测了肽给药到单克隆CTL群体的效果。为了证实在多克隆CTL级下皮下接种E1A-肽疫苗是否也诱导E1A-特异性CTL耐受性，向野生型(wt)C57BL/6小鼠注射E1A-肽(图5a和5b)或对照肽(图5c和5d)。一天后对这些小鼠用在其表面表达高水平E1A-肽的同源细胞系加强(图5b和5d)。将该细胞系注射到用对照肽引发的小鼠中容易地诱导E1A-特异性免疫(图5d)。但是，在注射该E1A/IFA疫苗之后小鼠封固E1A-特异性CTL反应的能力被废除(图5b)。这些数据说明，注射该E1A-肽不仅导致在TCR-转基因小鼠中而且在表达多克隆E1A-特异性T细胞所有组成成分的小鼠中的E1A-特异性耐受性。
由于皮下注射该E1A/IFA疫苗导致了全身CTL耐受性，因此研究了该E1A-肽是否分散全身并呈递到周边的前体CTL上。因此，对小鼠皮下注射乳化于IFA中的E1A-肽或来自人乳头瘤病毒(HPV)16 E7的对照肽。16个小时后从这些小鼠中分离脾细胞并将其用作体内E1A-特异性CTL克隆的刺激细胞。来自皮下注射有E1A-肽的小鼠的脾细胞诱导特异性繁殖，然而来自皮下注射有E7-肽的小鼠的脾细胞不能这样做(图6)。而且，对照CTL克隆在来自注射有E1A的小鼠的脾细胞上不繁殖(数据没有显示)。因此，这些数据说明，尤其通过脾细胞将在周边中全身地呈递皮下注射IFA中的E1A-肽。
从上面所述的E1A-肽疫苗的耐受性效果的角度来看，存在一问题，CD40在体内的触发是否足够地防止了CTL的周边耐受性并恢复了CTL引发。因此，研究了耐受性肽的注射与体内CD40触发一起是否能够防止导致肿瘤-特异性CTL免疫的周围CTL耐受性的诱导。
在实施例1和2中已显示了，体内CD40的触发能够代替引发辅助依赖性CTL反应时对CD4+T辅助细胞的需求。由于在防止周围CTL耐受性诱导时已包含CD4+T细胞所介导的辅助活性，因此本发明人解决了体内CD-40的触发是否足够防止周围E1A-特异性CTL的耐受性的问题。为此，对小鼠注射该E1A/IFA疫苗和活化抗-CD40 mAb FGK-45。接受这种组合的小鼠封固了强的E1A-特异性CTL反应(图7b和7e)，然而仅仅接受该E1A/IFA疫苗(图7e)或mAb的小鼠没有封固(未显示)。E1A/IFA疫苗和抗-CD40 mAb的组合在CD40-有缺陷的小鼠中不能引出CTL(图7c和7d)。而且，该E1A/IFA疫苗与同种型配对的对照mAb(图6a)或IL-2的共注射不能将该E1A/IFA疫苗诱导的CTL耐受性转变成CTL引发(未显示)。在仅用E1A-肽、或者E1A-肽加抗-CD40接种疫苗的动物中对该E1A-表位的反应的范围和差异显示在图7e中。这样，全身CD40的激活能够将肽所诱导的周围CTL耐受性倒转成肽和肿瘤-特异性CTL所介导的免疫。
E1A-特异性免疫的诱导与用含有E1A-肽的与PE共轭的H-2-Db-四聚体(Db/E1A)染色的接种疫苗的小鼠的脾脏中存在CD8+T细胞有很大关系。接种疫苗后10天内，通过流式细胞测量术能够在注射E1A-肽和抗-CD40 mAb的小鼠中检测出用Db/E1A四聚体染色的CD8+T细胞，但在仅注射E1A-肽的小鼠中不能检出(未显示)。在注射E1A-肽的小鼠中，用Db/E1A四聚体染色的CD8+细胞的百分比为约3％。在用整腺病毒(它诱导有效E1A-特异性免疫)接种疫苗的小鼠中，检测出可比较量的Db/E1A四聚体反应性CD8+脾脏细胞。这些结果说明，E1A-特异性CD8+T细胞在接受E1A/IFA疫苗和抗-CD40 mAb的小鼠中的扩展大致为或等于在接种病毒的动物中发现的。
实施例4：CD40的触发大大提高了肽基抗癌疫苗的功效
尽管上述的发现显示了通过CD40触发的帮助足够防止了给药耐受原肽疫苗之后CTL的耐受性，但是他们没有解决正常诱导保护性免疫而不是耐受性的抗癌疫苗的功效是否能通过CD40的激活得到提高。检测了体内CD40的触发对用来自HPV16 E7的肽的接种疫苗的结果是否有益。用这种肽接种疫苗将诱导抗用HPV16转化的肿瘤细胞刺激的保护性CTL所介导的免疫。而且，当该肽负载在体内激活的DC上时能够将该肽用于治疗调整，这暗示当该抗肿瘤反应通过激活的DC呈递时其强度得到加强。
与仅用E7-肽处理的小鼠(未显示)相比，接受E7-肽并伴随CD40触发的小鼠封固了更有效的CTL反应，这说明CD40触发也提高了HPV16 E7-肽疫苗的功效，并证实了具有上述E1A肽的发现。而且，经皮下注射仅带有HPV16 E7-肽(平头开口)的CD40-HPV16 E6/E7转化的肿瘤细胞处理6天后的小鼠能够使肿瘤生长减慢，但是最终大多数动物死于肿瘤(图8)。但是，当将HPV16 E7-肽接种疫苗与抗-CD40 mAb的注射相结合时，肿瘤的生长明显降低，并且10只小鼠中有7只没有排斥肿瘤，然而注射有对照肽和抗-CD40 mAb的动物不能控制肿瘤生长。这些结果显示了当免疫与体内CD40触发组合时能够明显提高接种机制的效果。这些数据为癌症患者提供了开发极有效并新颖的抗肿瘤疫苗的基础。
前面的描述、术语、解释和实施例仅为举例并不是限制性的。本发明包括前述实施方式的所有已知和未知的等价物。本发明仅通过以下的权利要求书而不是通过本文的任意其它部分或任意其它文献中的任意描述来限制。
序列表＜110＞考内里斯J.M.梅烈夫
瑞因克.欧夫林加
瑞内.托艾斯
史蒂芬P.斯考因伯格
马克.德.鲍尔＜120＞用于治疗肿瘤的CD40结合分子和CTL肽＜130＞98-4-pct＜150＞06/086,625＜151＞1998-05-23＜160＞3＜170＞FastSEQ for Windows Version 4.0＜210＞1＜211＞9＜212＞PRT＜213＞小鼠＜220＞＜400＞1Val Asn Ile Arg Asn Cys Cys Tyr Ile1 5＜210＞2＜211＞10＜212＞PRT＜213＞小鼠＜400＞2Ser Gly Pro Ser Asn Thr Pro Glu Ile1 5 10＜210＞3＜211＞9＜212＞PRT＜213＞乳头瘤病毒＜400＞3Arg Ala His Tyr Asn Ile Val Thr Phe1 5
Claims (13)
1.一种药物组合为，包括CD40结合分子和激活CTL的肽。
2.权利要求1的药物组合物，其中所述CD40结合分子为抗-CD40抗体或其片段、肽、低聚核苷酸或有机分子。
3.权利要求2的药物组合物，其中所述抗-CD40抗体为人、人化、嵌合或去免疫的。
4.权利要求1的药物组合物，其中所述激活CTL的肽为来自腺病毒且具有序列SGPSNTPPEI(SEQ ID NO：1)的E1A肽或来自人乳头瘤病毒16型且具有序列RAHYNIVTF(SEQ ID NO：3)的HPV16 E7肽。
5.一种治疗肿瘤的方法，包括给药权利要求1-4中任意的药物组合物。
6.一种治疗肿瘤或传染病的方法，包括给药CD40结合分子和激活CTL的肽。
7.权利要求5的方法，其中直接向肿瘤给药所述药物组合物。
8.一种治疗肿瘤或传染病的方法，包括给药编码CD40结合分子和激活CTL的肽的基因构建体。
9.权利要求8的方法，其中所述CD40结合分子为抗-CD40抗体或其片段、或肽，并且所述激活CTL的肽为来自腺病毒且具有序列SGPSNTPPEI(SEQ ID NO：2)的E1A肽或来自人乳头瘤病毒16型且具有序列RAHYNIVTF(SEQ ID NO：3)的HPV16 E7肽。
10.用权利要求8的基因构建体转染或感染的细胞。
11.权利要求8或9的方法，其中在体外或体内进行基因构建体的转染或感染。
12.权利要求11的方法，其中通过电穿孔、磷酸钙转染、微注射或者通过将所述基因构建体加入适宜脂质体中在体外进行所述转染。
13.权利要求12的方法，其中通过将所述基因构建体加入反转录病毒、腺病毒或细小病毒载体、或者通过将所述基因构建体或所述基因构建体与病毒或质粒载体加入适宜脂质体中在体内或体外进行所述感染。
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| US5225539A (en) | 1986-03-27 | 1993-07-06 | Medical Research Council | Recombinant altered antibodies and methods of making altered antibodies |
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| NZ273504A (en) * | 1993-10-01 | 1997-12-19 | Immunex Corp | Monoclonal antibodies (moabs) that specifically bind human cd40 molecules and cd40 ligand binding |
| NZ305083A (en) * | 1995-03-01 | 1999-06-29 | Immunex Corp | Use of cd40 binding proteins (a cd40 ligand or monoclonal antibody that specifically binds to cd40) for stimulating a immune response |
| AU738649B2 (en) * | 1996-04-26 | 2001-09-20 | Rijksuniversiteit Te Leiden | Methods for selecting and producing T cell peptide epitopes and vaccines incorporating said selected epitopes |
| DE69833755T2 (de) | 1997-05-21 | 2006-12-28 | Biovation Ltd. | Verfahren zur herstellung von nicht-immunogenen proteinen |
| WO1999061065A1 (en) * | 1998-05-23 | 1999-12-02 | Leiden University Medical Center | Cd40 binding molecules and ctl peptides for treating tumors |
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1999
- 1999-05-21 WO PCT/US1999/011419 patent/WO1999061065A1/en active IP Right Grant
- 1999-05-21 AT AT99937841T patent/ATE304375T1/de active
- 1999-05-21 AU AU43112/99A patent/AU765822B2/en not_active Ceased
- 1999-05-21 JP JP2000550524A patent/JP4927254B2/ja not_active Expired - Fee Related
- 1999-05-21 ES ES05107101T patent/ES2330017T3/es not_active Expired - Lifetime
- 1999-05-21 CN CNA2005100752729A patent/CN1762492A/zh active Pending
- 1999-05-21 CN CNB998076694A patent/CN1212155C/zh not_active Expired - Fee Related
- 1999-05-21 EP EP05107101A patent/EP1616579B1/en not_active Expired - Lifetime
- 1999-05-21 ES ES99937841T patent/ES2249907T3/es not_active Expired - Lifetime
- 1999-05-21 EP EP99937841A patent/EP1082141B1/en not_active Expired - Lifetime
- 1999-05-21 DK DK99937841T patent/DK1082141T3/da active
- 1999-05-21 NZ NZ508831A patent/NZ508831A/en not_active IP Right Cessation
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- 1999-05-21 DE DE69927262T patent/DE69927262T2/de not_active Expired - Lifetime
- 1999-05-21 DE DE69941165T patent/DE69941165D1/de not_active Expired - Lifetime
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2002
- 2002-08-26 US US10/227,789 patent/US7563445B2/en not_active Expired - Fee Related
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- 2009-07-17 US US12/505,022 patent/US20090285814A1/en not_active Abandoned
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| ES2249907T3 (es) | 2006-04-01 |
| DE69927262D1 (de) | 2006-01-26 |
| CN1762492A (zh) | 2006-04-26 |
| EP1082141A1 (en) | 2001-03-14 |
| EP1616579A1 (en) | 2006-01-18 |
| CN1212155C (zh) | 2005-07-27 |
| JP4927254B2 (ja) | 2012-05-09 |
| WO1999061065A1 (en) | 1999-12-02 |
| DE69941165D1 (de) | 2009-09-03 |
| AU765822B2 (en) | 2003-10-02 |
| NZ508831A (en) | 2004-08-27 |
| ATE304375T1 (de) | 2005-09-15 |
| US20090285814A1 (en) | 2009-11-19 |
| US7563445B2 (en) | 2009-07-21 |
| EP1082141B1 (en) | 2005-09-14 |
| US20030022860A1 (en) | 2003-01-30 |
| JP2002528383A (ja) | 2002-09-03 |
| DK1082141T3 (da) | 2005-12-05 |
| EP1616579B1 (en) | 2009-07-22 |
| EP1082141A4 (en) | 2002-08-07 |
| DE69927262T2 (de) | 2006-04-27 |
| ES2330017T3 (es) | 2009-12-03 |
| AU4311299A (en) | 1999-12-13 |
| DK1616579T3 (da) | 2009-09-14 |
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