CN103476458B - Toll样受体激动剂治疗癌症的用途 - Google Patents
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Abstract
本发明涉及用于通过提供Toll样受体激动剂,例如鞭毛蛋白,治疗表达Toll样受体5的组织中的癌症的方法和试剂。本发明还涉及使用Toll样受体激动剂保护肝脏免受肝毒性。
Description
技术领域
本发明涉及治疗表达Toll样受体的组织中的癌症的方法,并且涉及使用TLR激动剂保护肝脏免受肝毒性影响的方法。
发明背景
死亡受体家族及其同源配体之间的相互作用诱导控制组织,尤其是免疫系统中细胞群的内稳态的细胞凋亡。虽然许多肿瘤细胞类型对死亡配体敏感,但是Fas信号的活化也诱导了肝脏中的大量细胞凋亡,导致器官衰竭和死亡,妨碍其对系统性抗癌疗法的用途。Fas配体是在活化淋巴细胞和也可通过金属蛋白酶介导的裂解分泌出来并且以自分泌和旁分泌方式杀伤敏感细胞的许多肿瘤细胞上表达的Fas信号的40kDa生理激动剂。Fas是在活化淋巴细胞、多种组织和肿瘤细胞上表达的跨膜受体。Fas信号通过引起自分泌自杀或旁分泌死亡(细胞凋亡),通过消除活化淋巴细胞抑制免疫应答在免疫系统的调节中起至关重要的作用。结合后,Fas信号通过参与DISC形成的外部细胞凋亡通路、半胱天冬酶-8和10和内部(线粒体)细胞凋亡活化半胱天冬酶-8和Bid裂解和细胞色素释放诱导p53非依赖性细胞死亡。两种细胞凋亡通路导致半胱天冬酶-3和7活化。线粒体细胞凋亡受促凋亡和抗凋亡Bcl2家族膜调节。在肿瘤细胞中,在Fas受体不存在时,或NF-kB组成性活化,导致抗凋亡基因(例如c-Flip、Bcl-2、Bcl-xL)表达时,常常发现Fas信号调节异常。已经证明NF-kB反应性基因c-Flip抑制肿瘤中半胱天冬酶-8和Fas介导的细胞凋亡(REF,Kataoka等2000)。
发现通过Fas、TRAIL和TNFα死亡受体信号的p53非依赖性细胞凋亡机制后,因为肿瘤细胞通常p53功能受损,所以它们似乎是有希望的抗癌疗法目标。然而,死亡受体配体诱导严重的肝毒性。这阻碍了这些抗癌疗法的研发。虽然Fas激动剂引起肝损伤并且TNF-a在肝脏、肺和其它器官中引起严重炎症,但是在人体中TRAIL毒性最低。因此相比临床应用于抗癌疗法的其它激动剂,TRAIL受到更多的关注。然而,许多肿瘤对TRAIL疗法不敏感。当前采取了解决死亡受体毒性问题的几种方法,其中大部分旨在通过阻滞NF-kB活性和增加受体表达增强肿瘤敏感性,从而减少有效治疗所必需的药物量。另一个方向是使药物递送局部化于肿瘤以将对远处器官的毒性作用减到最小。迄今为止,没有预防毒性(包括肝损伤),允许在临床试验中全身应用死亡受体激动剂的可靠方法。因此,在本领域中需要在使用死亡受体治疗癌症时预防死亡受体的不期望的影响的方法。具体而言,需要保护肝脏免受这些不期望的影响。通常还需要保护肝脏免受肝毒性。
发现TLR在上皮和内皮细胞以及免疫细胞上表达。目前,在哺乳动物中已经鉴定了13种TLR。受体刺激后,几种常见信号通路被活化,例如NF-kB、AP-1、PI3K/AKT和有丝分裂原活化的蛋白激酶(MAPK),导致存活率升高,刺激细胞增殖和分泌许多具有趋化性和促炎功能的细胞因子。癌细胞中TLR的诱导可用于治疗癌症,然而,不同TLR的分布在各种器官和细胞类型中显著不同。这影响细胞因子谱和细胞炎症反应的程度。因此,在本领域中需要不依赖于TLR5表达存在的癌症免疫疗法。
发明概述
本文提供了一种治疗哺乳动物的癌症的方法,所述方法可包括向有需要的哺乳动物施用Toll样受体(TLR)激动剂。还提供了一种减少哺乳动物的癌症复发的方法,所述方法可包括向有需要的哺乳动物施用TLR激动剂。所述癌症可能存在于表达TLR的组织中。所述癌症可为转移或肿瘤再生。
TLR激动剂可为鞭毛蛋白。所述癌症可能不表达TLR,TLR可为TLR5。所述组织可为肝脏、肺、膀胱或肠道。所述癌症可是转移性的。所述癌症可为黑素瘤、结肠癌、乳腺癌、前列腺癌或恶性血液肿瘤,所述恶性血液肿瘤为淋巴瘤。所述癌症可为肿瘤。
所述激动剂可作为单一疗法施用。所述哺乳动物未接受联合治疗。所述哺乳动物也未接受化学治疗或放射治疗,但是可手术治疗。所述哺乳动物可具有足够的先天免疫力,所述先天免疫力可在相当于适合第一轮或后续化学治疗所需水平的水平上。所述哺乳动物的白细胞计数可在正常范围内,或白细胞计数可表明轻微免疫抑制。可在切除肿瘤之前、之后或同时向哺乳动物施用所述TLR激动剂。可在肿瘤切除期间施用所述TLR激动剂。
本文进一步提供了治疗哺乳动物的癌症的方法,所述方法可包括向有需要的哺乳动物施用FAS激动剂和TLR激动剂,所述TLR激动剂为鞭毛蛋白。所述FAS激动剂为FAS激动剂抗体。所述癌症是转移性的,并且可为肿瘤。所述癌症可能不表达TLR。所述癌症可能已经转移到表达TLR的被侵入组织。所述被侵入组织可为肝脏、膀胱、肺或肠道。
本文还提供了保护哺乳动物的肝脏组织免受肝毒性影响的方法,所述方法可包括向有需要的哺乳动物施用TLR激动剂。毒性可为FAS配体、FAS激动性抗体、TNFα、醋氨酚、醇、肝脏的病毒感染或化疗剂。毒性也可为沙门氏菌(Salmonella)感染,沙门氏菌感染可能来自鼠伤寒沙门氏菌(Salmonella typhimurium)。TLR激动剂可为鞭毛蛋白。
附图简述
图1示出了细菌鞭毛蛋白的结构域结构。Ca骨架跟踪、疏水核心分布和F41结构信息。限定结构域D1、D2a、D2b和D3的4个不同疏水核心。与Ca骨架一起展示了所有疏水侧链原子。侧链原子有色码:Ala,黄色;Leu、Ile或Val,橙色;Phe和Tyr,紫色(碳原子)和红色(氧原子)。c,鞭毛蛋白氨基酸序列中不同结构特征的位置和区域。从上到下,示出了:蓝色的F41片段;3个褐色的b-叶状折叠;具有黄色a-螺旋、绿色b-结构和紫色b-旋转的二级结构分布;每50个残基处蓝色的tic标记;结构域D0、D1、D2和D3;原型元件中青色的轴向亚基接触区域;红色的非常保守的氨基酸序列和紫罗兰色的可变区;F41中生成不同超螺旋元件的点突变。底部的字母指突变元件的形态:L(D107E、R124A、R124S、G426A),L型直链;R(A449V),R型直链;C(D313Y、A414V、A427V、N433D),卷曲33。
图2示出了沙门氏菌鞭毛蛋白结构域、其片段及其与TLR5的相互作用的示意图。黑色条形表示鞭毛蛋白基因用于构造包含A、B、C、A’和B’的片段的区域。
图3描绘了鞭毛蛋白衍生物。所选编码蛋白衍生物(列于右侧)的结构域结构和近似边界(氨基酸坐标)。在505个氨基酸(aa)内编码都柏林沙门氏菌(Salmonella dublin)的FliC鞭毛蛋白。
图4示出了下列鞭毛蛋白变体的核苷酸和氨基酸序列:AA’(SEQ ID NO:7-8)、AB’(SEQ ID NO:9-10)、BA’(SEQ ID NO:11-12)、BB’(SEQ ID NO:13-14)、CA’(SEQ ID NO:15-16)、CB’(SEQ ID NO:17-18)、A(SEQ ID NO:19-20)、B(SEQ ID NO:21-22)、C(SEQ ID NO:23-24)、GST-A’(SEQ ID NO:25-26)、GST-B’(SEQ ID NO:27-28)、AA’nl-170(SEQ ID NO:29-30)、AA’nl-163(SEQ ID NO:33-34)、AA’n54-170(SEQ ID NO:31-32)、AA’n54-163(SEQID NO:335-36)、AB’nl-170(SEQ ID NO:37-38)、AB’nl-163(SEQ ID NO:39-40)、AA’nl-129(SEQ ID NO:41-42)、AA’n54-129(SEQ ID NO:43-44)、AB’nl-129(SEQ ID NO:45-46)、AB’n54-129(SEQ ID NO:47-48)、AA’nl-100(SEQ ID NO:49-50)、AB’nl-100(SEQ ID NO:51-52)、AA’nl-70(SEQ ID NO:53-54)和AB’nl-70(SEQ ID NO:55-56)。用斜体字示出了pRSETb前导序列(前导序列包括Met,后者也是FliC的氨基酸1)。N端恒定结构域加有下划线。将氨基酸连接序列加粗。C端恒定结构域加有下划线。若存在GST,将其突出显示。
图5示出了对来自21种细菌的保守氨基(图5A)和羧基(图5B)末端的氨基酸序列的比较。用阴影示出了13个对于TLR5活性而言重要的保守氨基酸。用其登记号从TrEMBL(首字母=Q)或Swiss-Prot(首字母=P)中鉴定氨基酸序列。
图6示出了人TLR5的序列。
图7示出了响应于CBLB502和LPS注射体内NF-kB活化。A.在用CBLB502(0.2mg/kg)处理2h后,通过无损成像检测BALB/c-Tg(IκBα-luc)Xen报告小鼠体内的背景和NF-kB依赖性萤光素酶表达。B.皮下注射100μl PBS、CBLB502(0.2mg/kg)或LPS(1mg/kg)2h后,评估报告小鼠肝脏、小肠(回肠部分)、结肠、脾脏、肾脏、肺和心脏中的NF-kB依赖性萤光素酶表达。检测了每一组3只小鼠体内每μg蛋白质提取物的标准化萤光素酶活性。条形表示平均值+/-标准偏差。C.表示来自皮下注射了CBLB502或LPS的NIH-Swiss小鼠的肝脏中激动剂LPS和CBLB502生物活性的NF-kB核易位(p65)的动态。向对照小鼠注射PBS。在处理20、40和60min后获得组织样品,将其处理成石蜡块。在用CBLB502(100ng/ml)或LPS(1μg/ml)体外处理指定时间后检测从NIH-Swiss小鼠(D)分离的原代小鼠肝细胞和从(BD Biosciences)购买的人肝细胞(E)中p65的核易位。对照肝细胞保持完整。p65染绿色荧光,细胞角蛋白-8染红色荧光而细胞核染非特异性Dapi蓝染色。在放大×20倍下照相。箭头指基于形态标准确定的Kupffer和内皮细胞。
图8示出了CBLB502对Fas介导的肝毒性的防护。A.单独腹腔注射4μg抗Fas抗体或在抗体前30min、2h和6h组合注射CBLB502(1μg/只小鼠)后NIH-Swiss小鼠的的存活率。括号内是每次处理小鼠的数量。B.保护肝脏免受抗Fas抗体毒性。使用TUNEL技术检测注射抗Fas抗体5h后肝脏内的细胞凋亡。C.经苏木精伊红(H&E)染色的组织形态显示了注射抗Fas抗体对肝脏的坏死损害和CBLB502的保护。D.使用红细胞自身荧光(若丹明通道,红色)、小鼠IgG对照(Cy5偶联的抗小鼠IgG抗体,伪紫色)和DAPI细胞核(蓝色)检测肝脏出血。E.用或不用CBLB502预处理30min,注射3μg抗Fas抗体5h后在组织蛋白溶解产物中测定NIH-Swiss小鼠肝脏样品中的半胱天冬酶-3/7活性。N=3。条形表示平均值+/-标准偏差。F.用或不用CBLB502,抗Fas抗体注射5h后检测NIH-Swiss小鼠血清中的谷丙转氨酶(ALT)积聚。N=3。条形表示平均值+/-标准偏差。G.用或不用CBLB502预处理30min,注射3μg抗Fas抗体5h后在组织蛋白溶解产物中测定NIH-Swiss小鼠肝脏样品中的半胱天冬酶-8活性。N=3。条形表示平均值+/-标准偏差。
图9示出了CBLB502对肝脏中细胞凋亡相关因素的调节及其在CT-26肿瘤模型中对Fas介导的抗肿瘤活性的影响。通过蛋白质印迹检测单独或与CBLB502组合注射抗Fas抗体(5μg)2h后从C57BL/6小鼠分离的肝脏中CBLB502对半胱天冬酶-8(A)和Bid(B)裂解的抑制。C.通过RT-PCR检测用CBLB502处理30min和2h的完整小鼠的肝脏中Bcl2A1B、Bcl2A1D、IER-3、Fos、Jun和JunB基因的RNA表达。使用GAPDH作为对照以监测对基因表达的诱导。D.为具有皮下生长的CT-26肿瘤的小鼠注射单一抗Fas抗体(4μg/只小鼠)和CLB502或其组合。对照小鼠(“完整”)接受代替CBLB502的PBS和抗体。括号内是每一组中肿瘤的数量。结果表示平均肿瘤体积(m+/-标准误差)。(*)-完整和组合处理组之间的差异显著(p<0.05)。E.脾内注射表达萤光素酶的CT-26肿瘤细胞后第5天,用单独或与CBLB502组合的抗Fas抗体处理小鼠。在肿瘤细胞接种后第10、15、17、22、28和40天,使用Xenogen IVIS成像系统测定肝脏内的肿瘤生长。呈现了在第15天为来自每一组的3只小鼠获取的图像。在星号所示天数,CBLB502处理组和对照组中肝脏无肿瘤的小鼠比例之间的差异达到统计学显著(p<0.05)。F.用CBLB502处理5h后免疫细胞移动并浸润(箭头)至小鼠肝脏中生长的肿瘤节结中。
图10.注射CBLB502(5μg,皮下)或LPS(20μg,皮下)后,不同器官中NF-kB活化的动态。CO2吸入2、6、24和48h后使小鼠安乐死。将来自肝脏、大肠、肾脏和肺的蛋白质提取物中的萤光素酶活性标准化为每μg蛋白质提取物并且计算每个器官的平均值。按来自TLR激动剂处理小鼠的器官的蛋白质提取物中的平均萤光素酶活性和来自注射了PBS的对照小鼠的相应器官的提取物中获得的平均萤光素酶活性之间的比例计算萤光素酶诱导倍数(3只小鼠/组)。条形表示作为平均值±标准误差的诱导倍数。
图11.从萤光素酶报告小鼠分离并用CBLB502(100ng/ml)、LPS(5μg/ml)或PBS对照体外处理3h的小鼠肝细胞原代培养物中的NF-kB依赖性萤光素酶表达。然后用PBS冲洗肝细胞并收集于细胞溶解缓冲液(Promega)中。用Promega报告系统测定蛋白质上清液中的萤光素酶活性并标准化为每μg蛋白质提取物。条形表示萤光素酶单位(平均值±标准偏差)。
图12.在处理后的不同时间点获得的来自用CBLB502、抗Fas抗体(3μg)或其组合处理的NIH-Swiss小鼠的肝脏样品的苏木精伊红染色。在注射抗Fas抗体5、12和26h后获得肝脏样品,于10%福尔马林中固定,包埋于石蜡中并用苏木精和伊红染色用于组织形态。
图13.B16和CT-26细胞上的TLR5表达。A.通过RT-PCR测定B16和CT-26细胞中编码TLR5和GAPDH(作为对照)基因的mRNA的表达。使用对小鼠TLR5基因有特异性的引物来扩增小鼠TLR5mRNA的一个区域:正向(5’-AGTCCCCCAGCTCCAGTTTC-3’)和反向(5’-GGAGCCCCCTAGCAGTG AGT-3’)。B.使用萤光素酶报告基因测定测试B16和CT-26肿瘤细胞中响应于CBLB502、小鼠TNFα和LPS的NF-kB活化。数据表示萤光素酶单位(平均值±标准偏差)。
图14.示出了CT-26肿瘤细胞和A20淋巴瘤细胞中的TLR5表达。图14A和C,使用原稿正文中描述的TRIzol试剂从CT-26和B16肿瘤细胞(图14A)和CT-26和A20细胞(图14C)提取总RNA。使用LaserGene软件(DNASTAR,Inc.,Madison,WI)设计TLR5的引物。使用对小鼠TLR5基因有特异性的引物扩增小鼠TLR5mRNA的一个区域(GenBank登记号NM_016928.2):正向(5’-AGTCCCCCAGCTCCAGTTTC-3’)和反向(5’-GGAGCCCCCTAGCAGTGAGT-3’)。使用GAPDH作为对照以监测对基因表达的诱导。使用SuperscriptTM II逆转录酶和寡聚(dT)12-18引物(Invitrogen,Carlsbad,CA)合成cDNA。B.对B16(TLR5阳性)和CT-26TLR5阴性)肿瘤细胞中的NF-kB活化进行体外萤光素酶测定。
图15示出了CBLB502或PBS(未处理)处理(0.2mg/kg,皮下,1、2、3天)后,无胸腺裸鼠体内TLR5阳性HCT116肿瘤生长的动态,n=6-10。
图16示出了CBLB502或PBS(未处理)处理(0.2mg/kg,皮下,1、2、3天)后,无胸腺裸鼠体内的293-TLR5肿瘤生长,n=6-10。
图17示出了2个过程的CBLB502对比PBS(对照)处理(1、2、3、14、15和16天)期间,无胸腺裸鼠体内异种A549肿瘤生长的动态,n=6-10。无胸腺小鼠体内作为异种移植物在皮下生长的结肠HCT116腺癌的抗肿瘤活性。向8只无胸腺裸鼠的2个腹侧皮下注射HCT116(0.5×106个/100ml PBS)以诱导肿瘤。当肿瘤直径变成约3-5mm时(到注射后第6天),将小鼠随机分成2组,5只小鼠为CBLB502处理组而3只小鼠为PBS对照组。
图18示出了CBLB502或PBS(未处理)处理(0.1mg/kg,皮下,1、2、3天)后同源C3H小鼠体内SCCVII原位肿瘤生长的速率达到400mm3肿瘤尺寸,n=6-10。右图表示用和不用CBLB502处理,肿瘤达到400mm3体积所需的天数。
图19.经腹腔施用CBLB502(0.2mg/kg)3天,每天一次,处理具有皮下生长的同源Ward结肠肿瘤的Fischer大鼠。
图20示出了CBLB502与PBS(对照)处理(1、2、3天)后,无胸腺裸鼠体内异种A549-shV和A549-shTLR5肿瘤生长的动态。在第2、4、6和8天,在A549-shV肿瘤中观察到的肿瘤体积之间的统计差异(p<0.05),n=9-14。右图证明A549-shV和A549-shTLR5中响应于CBLB502处理NF-kB依赖性诱导萤光素酶报告基因表达。
图21示出了CBLB502与PBS(对照)处理(1、2、3天)后,无胸腺裸鼠体内H1299(对照)和H1299-TLR5肿瘤生长的动态,n=6。右图证明如H1299-TLR5细胞中的TLR5功能所表明,响应于CBLB502处理IL-8生成。
图22显示膀胱强烈响应于CBLB502。
图23显示CBLB502处理延迟了肝脏,甚至不表达TLR5的肿瘤中的肿瘤出现和生长。
图24示出了CBLB502对Fas介导的肝毒性的防护。
图25显示CBLB502保护肝脏免受TNFα和LPS毒性。
图26显示CBLB502保护肺免受TNF和LPS毒性。
图27显示CBLB502保护小鼠免于口服施用致死沙门氏菌。
图28显示伊立替康消除了鞭毛蛋白(CBLB502)的抗肿瘤作用。
发明详述
发明人已经得出了一个惊人的发现,甚至当细胞不表达TLR5时,提供Toll样受体(TLR)激动剂,例如TLR5的激动剂(如鞭毛蛋白),可有效抑制癌细胞的生长并减少癌细胞。TLR激动剂可能在治疗原发性或转移性肝癌、膀胱癌、肺癌和肠道癌,以及影响其它TLR5阳性组织的癌症中特别有用。TLR激动剂也可用于治疗在除肝脏、膀胱、肺、肠道和其它TLR5阳性组织以外的组织中产生,但是转移到这些组织的癌症。即使转移性癌细胞不表达TLR5,但是当癌症已经转移到表达TLR5的组织,例如肝脏时,也可用TLR激动剂治疗癌症。虽然不受理论约束,但是本发明实现的想法是,TLR激动剂有效减少或杀伤影响具有强大的先天免疫系统的组织的癌细胞,从而消除了对在癌细胞中任何预先存在的TLR5表达的需要。出乎意料地,通过提供TLR激动剂,充分触发先天免疫系统以便治疗缺乏TLR5表达的癌症。因此,为了TLR激动剂有效减少或杀伤癌细胞,不需要向癌细胞提供TLR5。
发明人还得出了惊人的发现,TLR激动剂可保护肝脏免受肝毒性。例如,FAS介导的细胞凋亡的死亡配体和活化剂(例如FAS配体和抗FAS激动性抗体)可诱导剂量依赖性肝毒性。施用TLR激动剂可保护肝脏免于此类毒性。TLR激动剂这种未预料到的性质允许其与FAS激动剂或TNF组合用于癌症治疗,以致减少或预防了FAS激动剂或TNF的副作用。
1.定义
本文使用的术语仅仅是为了描述特殊实施方案并非旨在限制。如说明书和所附权利要求中所使用,除非上下文另有明确指示,单数形式“一(a)”、“一(an)”和“所述(the)”包括复数指示物。
为了叙述本文的数值范围,明确涵盖了介于其间具有相同精确程度的介入数字。例如,对于6-9的范围而言,除6和9外还涵盖数值7和8,并且对于范围6.0-7.0而言,明确涵盖了数值6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9和7.0。
“施用”可指单剂量或多剂量的试剂。
在肽或多肽的情况下,“类似物”可指包含一个或多个非标准氨基酸或来自常规氨基酸集合的其它结构变型的肽或多肽。
“抗体”可指IgG、IgM、IgA、IgD或IgE类抗体或其片段或衍生物,包括Fab、F(ab’)2、Fd和单链抗体、双链抗体、双特异性抗体、双功能抗体及其衍生物。所述抗体可能是对所需表位或源自其中的序列表现出足够结合特异性的单克隆抗体、多克隆抗体、亲和纯化抗体或其混合物。所述抗体也可能是嵌合抗体。可通过连接本领域已知的一个或多个化学、肽或多肽部分衍生所述抗体。可使所述抗体与化学部分偶联。
“衍生物”可指一级结构(氨基酸和氨基酸类似物)不同的肽或多肽。衍生物的不同之处可能在于糖基化,一种翻译后修饰形式。例如,由于在异源系统中表达,肽或多肽可能表现出糖基化模式。如果保持至少一种生物学活性,则这些肽或多肽是根据本发明的衍生物。其它衍生物可包括具有共价修饰N-或C-末端的融合肽或融合多肽、PEG化肽或多肽、与脂质部分缔合的肽或多肽、烷基化肽或多肽、经氨基酸侧链官能团与其它肽、多肽或化学试剂连接的肽或多肽,并且另外的修饰如本领域所理解。
“片段”可指参考肽或多肽的一部分。
“同系物”可指共享一个共同的进化原种的肽或多肽。
“前导序列”可为编码与目标肽或多肽连接并与之一起翻译以使目标肽或多肽正确通过真核细胞内质网和高尔基复合体以便从细胞膜进行胞外分泌的任何肽序列的核酸。前导肽序列可源自碱性磷酸酶。前导序列可具有包含atgctgctgctgctgctgctgctgggcctgaggctacagctct ccctgggc的DNA序列。
“脂质体”可指由与细胞膜相同的材料制成的微小气泡(囊泡)。脂质体填充有药物并用于为癌症和其它疾病递送药物。脂质体可填充有载体。脂质体膜可由磷脂制成,磷脂是具有头基和尾基的分子。脂质体的头部可为水所吸引,而由长烃链构成的尾部为水所排斥。尾部可为水所排斥,并且排列起来远离水形成表面。质膜中的脂质可能主要是磷脂,如磷脂酰乙醇胺和磷脂酰胆碱。脂质体可由具有混合脂质链的自然衍生磷脂(如卵磷脂酰乙醇胺)或纯表面活性剂组分(如二油酰磷脂酰乙醇胺DOPE)构成。
“肽”或“多肽”可指连接的氨基酸序列并且可为天然、合成的或修饰或天然和合成的组合。
“大体上相同”可指第一和第二氨基酸序列在10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、200、300、400、500、600、700、800、900、1000、1100个氨基酸的区域上至少有60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%。
“治疗(treating)”、“治疗(treatment)”或“治疗(to treat)”各自可指在临时或永久基础上减轻、抑制、阻遏、消除、预防或减缓病状或病症的症状、临床体征或潜在病理的表现。预防病状或病症包括在疾病发作之前向受试者施用本发明的试剂。抑制病状或病症包括在诱导病状或病症之后,但是在其临床表现之前向受试者施用本发明的试剂。阻遏病状或病症包括在疾病的临床表现之后向受试者施用本发明的试剂。
“变体”可指因氨基酸的插入、缺失或保守性取代在氨基酸序列上不同,但是保持了至少一种生物学活性的肽或多肽。“生物学活性”的代表性实例包括与toll样受体结合和被特异性抗体结合的能力。变体也可指氨基酸序列与具有保持了至少一种生物学活性的氨基酸序列的参考蛋白大体相同的蛋白质。在本领域中认为氨基酸的保守性取代,即用具有相似性质(例如,亲水性、带电区域的程度和分布)的不同氨基酸置换氨基酸,通常涉及微小变化。如本领域中所理解,可通过考虑氨基酸的亲水指数部分来鉴定这些微小变化。Kyte等,J.Mol.Biol.157:105-132(1982)。氨基酸的亲水指数基于对其疏水性和电荷的考虑。本领域已知可取代具有相似亲水指数的氨基酸并且仍保持蛋白质功能。一方面,取代亲水指数为±2的氨基。氨基酸的亲水性也可用于揭示将产生保持生物学功能的蛋白质的取代。在肽的情况下,对氨基酸亲水性的考虑允许计算所述肽的最强局部平均亲水性,这是已经报道与抗原性和免疫原性非常相关的有用方法。美国专利No.4,554,101,通过引用全部并入本文。如本领域中所理解,取代具有相似亲水性值的氨基酸可产生保持生物学活性,例如免疫原性的肽。可用亲水性值在相互的±2以内的氨基酸进行取代。氨基酸的疏水性指数和亲水性值受该氨基酸的特殊侧链影响。与该观察情况一致,认为与生物学功能相容的氨基酸取代依赖于氨基酸,并且尤其是那些氨基酸的侧链的相对相似性,正如疏水性、亲水性、电荷、尺寸和其它性质所揭示的那样。
“载体”可指含有复制起点的核酸序列。载体可为质粒、酵母或哺乳动物人工染色体。载体可为RNA或DNA载体。载体可为自我复制的染色体外载体或整合至宿主基因组中的载体。
2.Toll样受体激动剂
本文提供了TLR激动剂。TLR激动剂可为PAMP,PAMP可能是源自病原体的保守分子产物。病原体可能是革兰氏阳性细菌、革兰氏阴性细菌、真菌或病毒。TLR激动剂可能是损伤相关分子模式(DAMP)配体,所述配体可能是从受损或垂死细胞释放的内源性分子。DAMP或PAMP可通过TLR信号发起免疫应答并且补充细胞质内的衔接分子以便传送信号。TLR激动剂可为可能是来自以下表1中的配体的TLR的激动剂:
表1TLR和配体
TLR激动剂可为结合TLR并诱导TLR介导的活性,例如活化NF-κB活性的PAMP或DAMP的片段、变体、类似物、同系物或衍生物。TLR激动剂片段、变体、类似物、同系物或衍生物可与TLR激动剂的氨基酸至少30-99%相同并且诱导TLR介导的活性。
TLR激动剂可靶向TLR,例如TLR-5。TLR激动剂可为TLR-5的激动剂并刺激TLR-5活性。TLR激动剂可为抗TLR5抗体或其它小分子。TLR激动剂可为鞭毛蛋白。
鞭毛蛋白也可能是鞭毛蛋白或鞭毛蛋白相关多肽。鞭毛蛋白可来自于任何来源,包括多种革兰氏阳性和革兰氏阴性细菌物种。鞭毛蛋白可能是来自革兰氏阳性或革兰氏阴性细菌物种的鞭毛蛋白多肽,包括但不限于美国专利公布No.2003/000044429中公开的鞭毛蛋白多肽,其内容通过引用全部并入本文。例如,鞭毛蛋白可具有来自美国专利公布No.2003/0044429的图7中描绘的细菌物种的氨基酸序列。在包括NCBI GenBank数据库的来源,编码美国2003/0044429的图7中所列鞭毛蛋白多肽的核苷酸序列公开可用。鞭毛蛋白也可能是与美国专利公布2003/000044429的图25中所示BLAST结果中所列的登记号相对应的鞭毛蛋白肽或其变体。鞭毛蛋白也可能是如美国专利申请公布No.2009/0011982中公开的鞭毛蛋白多肽,其内容全部并入本文。鞭毛蛋白可能是如本文图3和4中公开的任一种鞭毛蛋白多肽。
鞭毛蛋白可为结合TLR5并诱导TLR5介导的活性,例如活化NF-κB活性的鞭毛蛋白的片段、变体、类似物、同系物或衍生物。鞭毛蛋白的片段、变体、类似物、同系物或衍生物可与结合TLR5并且诱导TLR5介导的活性的鞭毛蛋白的氨基酸至少30-99%相同。
鞭毛蛋白可来自一种沙门氏菌,其代表性实例为都柏林沙门氏菌(由GenBank登记号M84972编码)。鞭毛蛋白相关多肽可为结合TLR5并诱导TLR5介导的活性,例如活化NF-kB活性的M84972片段、变体、类似物、同系物或衍生物或其组合。可根据鞭毛蛋白的结构域结构和TLR5识别的保守结构,通过基于合理的设计获得鞭毛蛋白的片段、变体、类似物、同系物或衍生物。
鞭毛蛋白可包含图2所示13个保守氨基酸中的至少10、11、12或13个(位置89、90、91、95、98、101、115、422、423、426、431、436和452)。鞭毛蛋白可与M84972的氨基酸1174和418505至少30-99%相同。其内容全部并入本文的美国专利申请公布No.2009/0011982的图26列出了具有已知TLR-5刺激活性的鞭毛蛋白的氨基和羧基末端与M84972相比的同一性百分比。
鞭毛蛋白可为细菌鞭毛的主要组分。鞭毛蛋白可由3个结构域构成(图1)。结构域1(D1)和结构域2(D2)可能不连续并且可在通过形成发夹结构使氨基末端和羧基末端的残基并列时形成。包含D1和D2结构域的氨基和羧基末端可能最保守,而中间高变结构域(D3)可能高度可变。对含有被大肠杆菌(Escherichia coli)铰链(ND1-2/ECH/CD2)分隔开的氨基D1和D2与羧基D1和D2的重组蛋白的研究表明,当与ECH元件偶联时,D1和D2可具生物活性。这种嵌合体(而不是单独的铰链)可诱导两个肠上皮细胞系中的IkBa降解、NF-kB活化及NO和IL-8生成。非保守D3结构域可能在鞭毛丝的表面并且可含有主要的抗原表位。鞭毛蛋白的强促炎活性可能存在于高度保守的N和CD1和D2区(见图1)。
鞭毛蛋白可通过与Toll样受体5(TLR5)结合诱导NF-kB活性。TLR可识别对于鞭毛蛋白而言特殊的保守结构。保守结构可由稍微允许氨基酸含量变化的一大群残基构成。Smith等,Nat Immunol.4:1247-53(2003)(其内容通过引用并入本文)已经鉴定了鞭毛蛋白中为TLR5识别的保守结构的一部分的13个保守氨基酸。图2中示出了鞭毛蛋白的对于TLR5活性而言可能很重要的13个保守氨基酸。
已经产生了保持至少一些TLR5刺激活性的鞭毛蛋白的大量缺失突变体。鞭毛蛋白可能是这种缺失突变体,并且可能是本文实施例中公开的缺失突变体。鞭毛蛋白可包含从缺少氨基酸185-306或444-492的GenBank登记号D13689或从缺少氨基酸179-415的GenBank登记号M84973翻译的序列或其变体。
鞭毛蛋白可包含转座子插入和对可变D3结构域的改变。可用允许D1和D2结构域正确折叠的铰链或连接子多肽部分或全部取代D3结构域,以致变体刺激TLR5活性。在大肠杆菌(E.coli)MukB蛋白中可找到变体铰链元件并且可具有2010年10月6日提交,其内容通过引用并入本文的国际申请No.PCT/US10/51646中提出的序列。
如上所述的鞭毛蛋白可进一步包含前导序列。进一步包含前导序列的鞭毛蛋白可为CBLB502S。
3.试剂
本发明还涉及包含治疗有效量的TLR激动剂的试剂。所述试剂可为多肽。所述试剂也可为载体。所述载体可包含编码TLR激动剂的核酸。所述载体可能能够转导哺乳动物细胞。可通过病毒或脂质体相关载体系统将载体递送至哺乳动物体内。病毒载体系统可为腺病毒或巨细胞病毒。
所述试剂可为携带载体的脂质体。所述脂质体可能能够转导哺乳动物细胞并递送载体进行表达。
所述试剂可为活化TLR的药物制剂,从而模仿大量渗透通过肠壁的情形使肿瘤或感染细胞暴露于宿主免疫系统。可于溶液中全身递送所述试剂,以便(例如)肌肉内施用。所述试剂可为表达呈纳米颗粒形式的TLR激动剂的药物制剂,所述纳米颗粒形式可将功能激动剂带到哺乳动物细胞的细胞表面。
所述试剂可为包含上述药物制剂的药物试剂,可使用本领域众所周知的方法生产所述药物试剂。所述试剂也可包含助剂。
所述载体可包含编码鞭毛蛋白的核酸。所述载体可能能够使用强启动子表达鞭毛蛋白。表达载体可进一步包含在编码TLR激动剂的基因上游克隆的前导序列。药物制剂可为表达以下的腺病毒:
于溶液中全身递送,以便(例如)肌肉内施用的TLR激动剂;或
表达呈携带功能TLR激动剂的纳米颗粒形式的TLR激动剂,例如可源自CBLB502表面的鞭毛蛋白。纳米颗粒可基于噬菌体T7,或全部形成为保持其生物学活性。纳米制剂可提供剂量依赖性、NF-κB反应性报告基因活化,并且可通过胞吞作用导致细胞内化以便有效免疫(Mobian AP-A)。
a.施用
可使用本文所述方法经全身、口服、肠胃外、舌下、经皮、直肠、经黏膜、局部、经吸入、经颊或其组合施用所述试剂。肠胃外施用包括但不限于静脉内、动脉内、腹膜内、皮下、肌肉内、鞘内和关节内。也可皮下、静脉内、经气管内或瘤内施用。对于兽用,可根据正常兽医实践作为可适当接受的制剂施用所述试剂。兽医可容易地确定给药方案和最适于特定动物的施用途径。可向人类患者、猫、犬、大型动物或禽类施用所述试剂。
所述试剂可作为单一疗法施用或与可为手术或切除肿瘤的其它治疗一起同时或有节奏地(metronomically)施用。如本文所使用,术语“同时(simultaneous)”或“同时(simultaneously)”指所述试剂和其它治疗可相互在48h,优选24h,更优选12h,还更优选6h和最优选3h或更少时间内施用。如本文所使用,术语“有节奏地”指在不同于其它治疗的时间并且按相对于重复施用的一定频率施用所述试剂。
可在另一治疗之前的任何点施用所述试剂,包括约120h、118h、116h、114h、112h、110h、108h、106h、104h、102h、100h、98h、96h、94h、92h、90h、88h、86h、84h、82h、80h、78h、76h、74h、72h、70h、68h、66h、64h、62h、60h、58h、56h、54h、52h、50h、48h、46h、44h、42h、40h、38h、36h、34h、32h、30h、28h、26h、24h、22h、20h、18h、16h、14h、12h、10h、8h、6h、4h、3h、2h、1h、55min、50min、45min、40min、35min、30min、25min、20min、15min、10min、9min、8min、7min、6min、5min、4min、3min、2min和1min。可在第二次试剂治疗之前的任何点施用所述试剂,包括约120h、118h、116h、114h、112h、110h、108h、106h、104h、102h、100h、98h、96h、94h、92h、90h、88h、86h、84h、82h、80h、78h、76h、74h、72h、70h、68h、66h、64h、62h、60h、58h、56h、54h、52h、50h、48h、46h、44h、42h、40h、38h、36h、34h、32h、30h、28h、26h、24h、22h、20h、18h、16h、14h、12h、10h、8h、6h、4h、3h、2h、1h、55min、50min、45min、40min、35min、30min、25min、20min、15min、10min、9min、8min、7min、6min、5min、4min、3min、2min和1min。
可在另一治疗之后的任何点施用所述试剂,包括约1min、2min、3min、4min、5min、6min、7min、8min、9min、10min、15min、20min、25min、30min、35min、40min、45min、50min、55min、1h、2h、3h、4h、6h、8h、10h、12h、14h、16h、18h、20h、22h、24h、26h、28h、30h、32h、34h、36h、38h、40h、42h、44h、46h、48h、50h、52h、54h、56h、58h、60h、62h、64h、66h、68h、70h、72h、74h、76h、78h、80h、82h、84h、86h、88h、90h、92h、94h、96h、98h、100h、102h、104h、106h、108h、110h、112h、114h、116h、118h和120h。可在第二次试剂治疗之后的任何点施用所述试剂,包括约120h、118h、116h、114h、112h、110h、108h、106h、104h、102h、100h、98h、96h、94h、92h、90h、88h、86h、84h、82h、80h、78h、76h、74h、72h、70h、68h、66h、64h、62h、60h、58h、56h、54h、52h、50h、48h、46h、44h、42h、40h、38h、36h、34h、32h、30h、28h、26h、24h、22h、20h、18h、16h、14h、12h、10h、8h、6h、4h、3h、2h、1h、55min、50min、45min、40min、35min、30min、25min、20min、15min、10min、9min、8min、7min、6min、5min、4min、3min、2min和1min。
b.制剂
所述方法可包括施用所述试剂。本文提供的试剂可呈以常规方式配制的片剂或锭剂形式。例如,供口服的片剂和胶囊可含有可能为粘合剂、填料、润滑剂、崩解剂和湿润剂的常规赋形剂。粘合剂包括但不限于糖浆、阿拉伯树胶、明胶、山梨糖醇、黄蓍胶、淀粉胶浆和聚乙烯吡咯烷酮。填料可为乳糖、食用糖(sugar)、微晶纤维素、玉米淀粉、磷酸钙和山梨糖醇。润滑剂包括但不限于硬脂酸镁、硬脂酸、滑石、聚乙二醇和二氧化硅。崩解剂可为马铃薯淀粉和羟基乙酸淀粉钠。湿润剂可为硫酸月桂酯钠。可根据本领域众所周知的方法对片剂进行包衣。
本文提供的试剂也可为液体制剂,例如水或油混悬液、溶液、乳液、糖浆和酏剂。也可将所述试剂配制成干品,以在使用之前用水或其它适合媒介物溶解。此类液体制剂可含有添加剂,例如助悬剂、乳化剂、非水媒介物和防腐剂。助悬剂可为山梨糖醇糖浆、甲基纤维素、葡萄糖/食用糖浆、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶和氢化食用脂肪。乳化剂可为卵磷脂、脱水山梨糖醇单油酸酯和阿拉伯树胶。非水媒介物可为食用油、扁桃仁油、分馏椰子油、油酯、丙二醇和乙醇。防腐剂可为对羟基苯甲酸甲酯或丙酯和山梨酸。
也可将本文提供的试剂配制成栓剂,其可含有栓剂基体,例如可可油或甘油酯。也可配制本文提供的试剂用于吸入,可呈例如可作为干粉施用的溶液、混悬液或乳液形式或呈使用推进剂,例如二氯二氟甲烷或三氯氟甲烷施用的气雾剂形式。也可将本文提供的试剂配制成包含含水或非水媒介物的透皮制剂,例如乳膏、软膏、洗剂、糊剂、膏药、贴片或膜。
也可配制本文提供的试剂用于肠胃外施用,例如通过注射、瘤内注射或持续输注。供注射的制剂可于含水媒介物中呈混悬液、溶液或乳液形式,并且可含有配制试剂,包括但不限于助悬剂、稳定剂和分散剂。也可提供呈粉末形式的试剂,以用适合媒介物溶解,包括但不限于无菌、无热原水。
也可将本文提供的试剂配制成可通过植入或肌肉注射施用的贮库制剂。可用适合的聚合或疏水性材料(例如,于可接受的油中呈乳液)、离子交换树脂或微溶衍生物(例如,呈微溶盐)配制试剂。
c.剂量
所述方法可包括向有需要的患者施用治疗有效量的试剂。治疗使用所需的治疗有效量随治疗病状的性质、活化TLR活性所需的时间长度和患者的年龄/状况而变化。然而,一般而言,用于成人治疗的剂量通常在每天0.001mg/kg至约200mg/kg的范围内。剂量可为每天约1mg/kg至约100mg/kg。所需剂量可方便地呈单剂量施用,或以适当间隔作为多剂量施用,例如每天2、3、4个或更多个子剂量。可能希望或需要多剂量。
所述剂量可为任何剂量,例如约0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、0.6mg/kg、0.7mg/kg、0.8mg/kg、0.9mg/kg、1mg/kg、25mg/kg、50mg/kg、75mg/kg、100mg/kg、125mg/kg、150mg/kg、175mg/kg、200mg/kg、225mg/kg、250mg/kg、275mg/kg、300mg/kg、325mg/kg、350mg/kg、375mg/kg、400mg/kg、425mg/kg、450mg/kg、475mg/kg、500mg/kg、525mg/kg、550mg/kg、575mg/kg、600mg/kg、625mg/kg、650mg/kg、675mg/kg、700mg/kg、725mg/kg、750mg/kg、775mg/kg、800mg/kg、825mg/kg、850mg/kg、875mg/kg、900mg/kg、925mg/kg、950mg/kg、975mg/kg或1mg/kg。
d.单一疗法
所述试剂可作为单一疗法施用,在单一疗法下,不与任何其它类型的癌症治疗,例如化学治疗、放射治疗、另一种生物治疗或其它联合治疗一起施用所述试剂;条件是“单一疗法”可包括与手术治疗一起施用所述试剂。可与手术组合施用所述试剂,手术可为肿瘤切除。可在手术之前、同时或之后施用所述试剂。可在手术期间施用所述试剂。
4.治疗癌症的方法
本文提供了通过向有需要的哺乳动物施用所述试剂,治疗可能存在于表达TLR(例如TLR5)的组织中的癌症的方法。所述癌症可能是肿瘤或转移性癌症。所述癌症也可能存在于肝脏、膀胱、肺或肠道组织中,并且也可能起源于另一类型的组织,例如结肠、乳腺或前列腺。所述癌症也可能是黑素瘤或恶性血液肿瘤,例如淋巴瘤。所述癌症也可能是已经转移到表达TLR的组织,例如肝脏、肺、膀胱、肠道或其它表达TLR的组织的任何癌症。所述癌症可能是TLR阴性癌,因此缺乏Toll样受体的表达。所述癌症可能缺乏Toll样受体的内源和外源表达两者。所述方法可包括不向癌症提供Toll样受体的步骤,其可包括不在外源或内源提供Toll样受体。所述癌症可能缺乏任何及所有Toll样受体表达。
a.Toll样受体
Toll样受体(TLR)可识别源自病原体的保守分子产物,但是可区别于宿主分子的分子,统称为病原体相关分子模式(PAMP),所述病原体包括革兰氏阳性细菌、革兰氏阴性细菌、真菌和病毒。TLR也可识别从受损或垂死细胞释放的内源性分子,统称为损伤相关分子模式(DAMP)。如以下进一步描述,PAMP或DAMP可为TLR激动剂。TLR可为补充细胞质内的衔接分子以便传送信号的片段、变体、类似物、同系物或衍生物。TLR可来自人类或其它哺乳动物物种,例如猕猴、小鼠或大鼠。TLR可与补充细胞质内的衔接分子的TLR至少30-99%相同,以便传送信号。
TLR可能是估计存在于大多数哺乳动物物种中的10-15个类型的TLR之一。TLR可能是在人类和小鼠中已经鉴定的13种TLR(简称TLR1-TLR13)之一,或可为在其它哺乳动物物种中已经发现的等效形式。TLR可能是在人类中已经鉴定的11个成员(TLR1-TLR11)之一。
TLR一般可由不同类型的免疫细胞表达,并且可位于细胞表面或细胞质内。TLR一般可在癌细胞上表达。TLR一般可由消化系统中的正常上皮细胞、皮肤中的正常角化细胞、肺泡和支气管上皮细胞及雌性生殖道的上皮细胞表达。器官内衬的这些细胞可能是防御微生物侵入的第一道防线,并且一般在上皮细胞中表达的TLR在对增殖和细胞凋亡的调节中可能有至关重要的作用。
TLR可能不由癌细胞表达。TLR阴性癌细胞可能不表达任何TLR mRNA,可能不表达任何TLR蛋白,或可能不表达任何功能TLR蛋白。由于结合TLR配体的能力减弱或传送通过配体结合触发的下游信号的能力减弱,TLR蛋白可能不起作用。TLR阴性癌细胞的TLR mRNA、蛋白质或TLR功能水平也可能降低。与来自产生癌症的组织的正常细胞相比,或与其它已知的表达TLR的细胞类型相比,可能降低100%,或大于99.9%、99%、95%、90%、85%、80%、75%、70%、65%、60%、55%或50%。表达TLR的细胞可为正常细胞或肿瘤细胞,例如肿瘤细胞系或肿瘤异种移植物。
一般在癌细胞上表达的TLR可上调NF-κB级联并生成有助于致癌和癌细胞增殖的抗凋亡蛋白。
已知在信号中涉及TLR的4种衔接分子。将这些蛋白质称为髓样分化因子88(MyD88)、Tirap(也称为Mal)、Trif和Tram。衔接因子活化细胞中的其它分子,包括放大信号的某些蛋白激酶(IRAK1、IRAK4、TBK1和IKKi),并最终导致对配合炎症反应的基因的诱导和抑制。病原体识别期间的TLR信号通路可通过MyD88、活化B细胞(NF-κB)的核因子κ轻链增强子和有丝分裂原相关蛋白激酶(MAPK)介导的细胞外和细胞内通路诱导免疫应答。总之,TLR信号活化了成千上万个基因,并且总的来说,TLR构成了基因调控最具多效性,然而严格调节的通路之一。
TLR与白细胞介素-1受体一起形成受体超家族,称为“白细胞介素-1受体/Toll样受体超家族”。这个家族所有成员的共同之处是具有称为TIR(Toll-IL-1受体)的结构域。可能存在TIR结构域的3个亚类。具有亚类I TIR结构域的蛋白质是巨噬细胞、单核细胞和树突细胞生成的白细胞介素的受体并且全部具有细胞外免疫球蛋白(Ig)结构域。具有亚类IITIR结构域的蛋白质是传统TLR,并且直接或间接与微生物来源的分子结合。含有TIR结构域(III)的第3亚类蛋白由全部为细胞溶质并介导来自亚类1和2蛋白质的信号的衔接蛋白组成。TLR可能是保持亚类I TIR结构域、亚类II TIR结构域或亚类III TIR结构域的片段、变体、类似物、同系物或衍生物。
TLR可起二聚体的作用。例如,虽然多数TLR似乎起同型二聚体的作用,但是TLR2与TLR1或TLR6形成异二聚体,每个二聚体具有不同配体特异性。TLR也可依赖于其它辅助受体以便对全部配体敏感,例如在TLR4识别LPS的情况下,需要MD-2。已知CD14和LPS结合蛋白(LBP)促进LPS向MD-2的呈递。
(1)TLR1
TLR可为TLR1,其以对革兰氏阳性细菌的特异性识别PAMP。TLR1也称为CD281。
(2)TLR5
TLR可为Toll样受体5。TLR5编码的蛋白质可能在病原体识别和先天免疫活化中起重要作用。TLR5可识别在传染剂上表达的PAMP并介导生成有效免疫发展所必需的细胞因子。TLR5可识别细菌鞭毛蛋白,后者是细菌鞭毛的主要成分和毒力因子。TLR5的活化可使核因子NF-κB移动并刺激肿瘤坏死因子-α生成。
(3)癌症类型
癌症可为原发性癌或转移性癌。原发性癌可为变得在临床上可检测的起源部位的一个区域的癌细胞,并且可为原发性肿瘤。相反,转移性癌可为疾病从一个器官或部分传播到另一非相邻器官或部分。转移性癌可由获得穿透并浸润局部区域中的周围正常组织的能力,形成可局部转移的新肿瘤的癌细胞引起。
转移性癌也可由获得穿透淋巴管壁和/或血管壁的能力的癌细胞引起,之后癌细胞能够通过血液循环(从而称为循环肿瘤细胞)至体内其它部位和组织。转移性癌可能是由于例如淋巴或血液传播的过程。转移性癌也可由停靠在另一部位上,再次穿透血管或壁,持续繁殖,并最终形成另一临床上可检测的肿瘤的肿瘤细胞引起。转移性癌可为这种可为转移性(或继发性)肿瘤的新型肿瘤。
转移性癌可由已经转移的肿瘤细胞引起,可为继发性或转移性肿瘤。转移性肿瘤细胞可能和原肿瘤中的细胞相同。例如,如果乳腺癌或结肠癌转移到肝脏,当存在于肝脏中时,继发性肿瘤由异常乳腺或结肠细胞,而非异常肝脏细胞构成。因此,肝脏中的肿瘤可为转移性乳腺癌或转移性结肠癌,而非肝癌。
转移性癌可能起源于任何组织。转移性癌可能源自黑素瘤、结肠、乳腺或前列腺,并且因此可由最初分别为皮肤、结肠、乳腺或前列腺的细胞构成。转移性癌也可以是恶性血液肿瘤(可能是淋巴瘤)。转移性癌可侵害组织,例如肝脏、肺、膀胱或肠道。被侵入组织可表达TLR,而转移性癌可能表达或不表达TLR。
b.组合
所述方法也可包括联合施用TLR激动剂和抗癌疗法。抗癌疗法可为FAS配体、FAS激动性抗体、TNFα、TNFα激动性抗体、TRAIL或TRAIL激动性抗体。TLR5激动剂可用于使得所述癌症对抗癌疗法敏感。所述方法也可与治疗癌症的其它方法组合,包括使用免疫刺激剂、细胞因子或化学治疗剂。免疫刺激剂可为生长激素、催乳激素或维生素D。
5.减少癌症复发的方法
本文提供了一种减少癌症复发的方法,包括向有需要的哺乳动物施用TLR激动剂。所述癌症可能或可能已经存在于表达或不表达TLR,例如TLR5的组织中。所述癌症、组织、TLR、哺乳动物和试剂可如上所述。所述方法也可防止癌症复发。所述癌症可为肿瘤疾病。
所述癌症可为休眠肿瘤,其可由癌症的转移引起。休眠肿瘤也可能是手术切除肿瘤所遗留下的。癌症复发可能是肿瘤再生、肺转移或肝脏转移。
6.哺乳动物
哺乳动物可能具有功能完整的免疫系统,并且不可能免疫受损。哺乳动物的免疫水平也可能与足以使得哺乳动物适合第一轮或第二轮化学治疗的水平相当。哺乳动物可能不具有可能由化学治疗引起的低白细胞计数。低白细胞计数可能是由化学治疗期间健康细胞损失引起。所述损失是化学治疗药物的预期副作用。低白细胞计数可能是化学治疗引起的严重免疫抑制。低白细胞计数可损害所述试剂的抗肿瘤作用。低白细胞计数可在化学治疗7-14天后恢复。
哺乳动物的白细胞计数可能在正常范围内。哺乳动物的白细胞计数也可能表明轻微免疫抑制。哺乳动物可能尚未接受7-14天,或至少14天的化学治疗。哺乳动物的总白细胞计数也可为至少3000或3500个细胞/ml全血;粒细胞计数为至少1800或2100个细胞/ml全血;或白蛋白水平为至少3.0或3.5g/100ml全血。白细胞计数、粒细胞计数或白蛋白水平也可能在这些水平的+/-5%、10%、20%、30%、40%或50%内。
7.保护肝脏的方法
如以上所讨论,通过FAS、TRAIL和TNFα死亡受体信号(例如死亡配体)触发细胞凋亡的抗癌疗法可引起严重的肝毒性。因此,已经限制使用分子,例如FAS、TRAIL和TNFα作为抗癌疗法,即使这些分子在靶向癌细胞中有效。因此,本文还提供了一种保护哺乳动物的肝脏组织免受肝毒性影响的方法。通过向哺乳动物施用所述试剂保护肝脏。死亡受体信号激动剂可为FAS、TRAIL或TNFα。死亡配体可为肝毒性。FAS、TRAIL或TNFα可用作抗癌剂。
肝毒性也可为沙门氏菌感染,沙门氏菌感染可能来自鼠伤寒沙门氏菌。所述试剂也可用于防御可能是FAS介导的肝毒性。毒性也可为FAS配体、FAS激动性抗体、TNFα、醋氨酚、醇、肝脏的病毒感染或化疗剂。可向哺乳动物施用所述试剂。
实施例1
TLR5激动剂保护肝脏免受肝毒性
由于至少部分抑制放射敏感组织中的细胞凋亡,药学上优化的TLR5激动剂CBLB502是一种有效的放射防护剂。试验CBLB502保护肝脏免受Fas介导的细胞凋亡。下列实施例证明,用CBLB502刺激后,小鼠和人类肝细胞中TLR5通路活化,导致NF-kB依赖性诱导编码抗凋亡蛋白的基因。用CBLB502预处理小鼠保护其免于致死剂量的Fas激动性抗体,减少Fas诱导的血液中肝酶、肝脏提取物中的半胱天冬酶活性升高并保护肝脏组织完整性。在同源黑素瘤和结肠癌小鼠模型中CBLB502并不保护肿瘤。这些观察情况支持在TLR5激动剂,例如CBLB502的保护下,使用Fas激动剂进行癌症治疗。
施用TLR5激动剂CBLB502和TLR4激动剂LPS(另一已知的NF-kB活化剂)后,比较不同器官中的NF-kB反应。发现CBLB502诱导肝细胞中NF-kB的迅速直接活化,而通过不同类型的细胞介导肝细胞中NF-kB的LPS活化。因此下列数据还证明,用CBLB502预处理可降低抗癌疗法期间小鼠体内Fas介导的肝毒性。以下描述的方法基于通过源自鼠伤寒沙门氏菌鞭毛蛋白的toll样受体-5(TLR5)激动剂CBLB502活化NF-kB增强正常组织对破坏性副作用的抗性。
1.响应于TLR4和TLR5激动剂体内NF-k活化的确定
与通过TLR4作用的细菌LPS比较,研究在小鼠不同器官中对TLR5激动剂CBLB502的NF-kB反应。NF-kB依赖性萤光素酶报告基因Xenogen小鼠模型,其中萤光素酶转基因在IkBα基因的NFkB依赖性天然启动子的控制下表达(Zhang N等,2005)。施用NFkB活化剂后,在对指定试剂起反应的细胞和组织中萤光素酶活性增强。使用非侵入性Xenogen成像系统和离体萤光素酶报告基因测定,在CBLB502皮下注射2h后检测小鼠肝脏中NF-kB强烈活化(图7A)。对不同器官中NF-kB活化的定量分析揭示,与LPS相比,CBLB502诱导肝脏中更强烈的NF-kB活化,肠道中同样高的NF-kB活化水平,而在脾脏、骨髓、肾脏和肺中发现NF-kB活性较低(图7B)。对于两种TLR激动剂而言,NF-kB诱导的萤光素酶报告基因活性的动态相似,注射约2h后活化谱达到峰值,在6h时间点降低并且在注射24h后不可有效检测(图10)。
对小鼠肝脏样品向核的p65易位的免疫组织化学染色揭示,早在注射20min后CBLB502直接活化肝细胞中的NF-kB,尚无Kupffer和内皮细胞反应(图7C)。CBLB502注射1h时,所有肝细胞,包括Kupffer细胞和内皮细胞,证明了p65的核积聚,这表明原发和继发性作用与旁分泌机制对NF-kB的后续活化重叠。相反,肝细胞中LPS活化的NF-kB核易位发生明显更迟。LPS施用约1h后首先在库普弗(Kupffer)细胞和内皮细胞中,接着在肝细胞中观察到NF-kB活化。
用CBLB502,而不用LPS处理原代肝细胞培养物(鼠和人)证明NF-kB易位至核(图7D、E)。用鼠肝细胞培养物,通过NF-kB依赖性萤光素酶表达确认CBLB502介导NF-kB活化,而LPS不诱导该细胞中的NF-kB活化(图11)。在LPS处理的肝细胞中发现NF-kB活化水平低更有可能是由于其它肝基质细胞污染了原代肝细胞培养物。
这些结果显示,肝细胞表达TLR5,但是不表达TLR4,使得CBLB502直接活化肝细胞中的NF-kB,而LPS最初活化其它细胞类型(免疫和/或基质)并且稍后仅作为继发事件,间接活化肝细胞。
2.CBLB502防护Fas介导的肝毒性
与已经证明的一样,抗Fas抗体可诱导剂量依赖性肝毒性并通过诱导细胞凋亡、肝组织坏死和出血迅速杀伤小鼠(Ogasawara J等,Nature1993,Nishimura等1997)。因此,TLR5激动剂CBLB502诱导的肝细胞中的NF-kB活化可保护肝脏免于Fas介导的细胞凋亡。在NIH-Swiss小鼠中,腹腔注射的4μg抗Fas抗体(克隆Jo2)诱导肝脏中大量细胞凋亡、坏死和出血(图8B、C和D),在抗体注射后的前1-2天内杀死小鼠(图8A)。与完整对照小鼠相比,在动力学上对用CBLB502处理的小鼠的病理形态学检查显示,其肝脏有轻微的肝细胞空泡形成(图12)。在动力学上对注射了亚致死剂量的抗Fas抗体(3μg/只小鼠)的检查揭示,与末端(中间)静脉相邻的细胞受更好保护的肝门通道周围的肝细胞明显凋亡,在5h时最明显并且随时间推移(注射12和24h后)减弱。在用CBLB502和抗Fas抗体处理的小鼠的肝脏中,变化最小并且肝细胞看上去接近正常-仅可见到轻微空泡形成和单个凋亡细胞。
当在抗Fas抗体前30min注射时,CBLB502注射小鼠对注射后更好总体存活率有偏差的肝脏损伤比NIH-Swiss小鼠低约80%(图8A)。当在抗体前2h注射CBLB502时,所有小鼠均存活。然后到预处理的6h时间点,保护水平降低。
2和3μg的抗Fas抗体仅诱导NIH-Swiss小鼠体内的瞬时肝毒性、肝脏中的半胱天冬酶3/7活化和血液中的谷丙转氨酶(ALT)分泌(图8E、F)。两次试验表明,如果用CBLB502预处理小鼠,则抗Fas抗体诱导的肝损伤显著降低。有趣的是,Balb/c和C57B1/6小鼠似乎对抗Fas抗体比NIH-Swiss小鼠敏感度低。4μg的抗Fas抗体(NIH-Swiss小鼠的致死剂量)仅在BALB/c和C57B1/6小鼠体内诱导瞬时半胱天冬酶3/7活化,这通过在抗体前30min注射CBLB502成功预防(图13)。
这些数据支持肝细胞中TLR5介导的NF-kB活化可为对Fas介导的毒性的抗性增强的指示和测量的假设。
3.肝脏中CBLB502抑制促凋亡并诱导抗凋亡因子
半胱天冬酶3和7是内部(线粒体)和外部(半胱天冬酶)Fas介导的凋亡信号的下游靶标。受体活化后,首先半胱天冬酶-8变得磷酸化并且裂解,导致通过裂解促凋亡Bid蛋白和细胞色素释放作用的线粒体凋亡机制活化(Lou等1998)。因此,我们检查CBLB502是否抑制这种机制。
对于半胱天冬酶-8和Bid的肝脏蛋白提取物的蛋白质印迹分析证明,与单一注射抗Fas抗体相比,在注射CBLB502和抗Fas抗体组合的小鼠体内,这些蛋白质裂解少得多(图9A、B)。一致地,正如使用荧光底物测定所表明,半胱天冬酶8活化降低至背景水平(图8F)。
CBLB502使小鼠免受Fas介导的肝毒性的保护随时间推移而增强,在30min-2h达到最大峰值的事实表明,在肝细胞中存在预处理事件。在许多细胞因子和抗凋亡因子中,通过RT-PCR确认,在CBLB502施用30min和2h后,通过RNA阵列杂交在肝脏中发现两个抗凋亡bcl2家族成员bcl2A1B和bcl2A1D上调(Chao和Korsmeyer,1998,Arikawa等2006)(图9C)。CBLB502也迅速诱导另一种抗凋亡蛋白,立即早期反应蛋白IER-3的RNA表达(图9C,IEX-1是替代名称),经证实所述蛋白抑制生成活性氧族和线粒体凋亡通路(Shen等2009)。对肝脏样品的RT-PCR分析揭示,施用30min后CBLB502已经诱导IER-3RNA表达,到2h时显著增强。发现MAPK通路的几种蛋白质在用CBLB502处理的小鼠的肝脏中上调。证明肿瘤中MAPK通路的活化介导了这些细胞对Fas受体凋亡(REF)的抗性。抗Fas抗体注射,接着用CBLB502预处理后,Jun、Jun-B和Fos基因表达的上调与小鼠存活率直接相关,这表明了它们在CBLB502介导的对Fas肝毒性的防御中的可能作用。
4.CBLB502对Fas介导的抗肿瘤活性的影响
LPS不是临床应用很好的候选,因为它在许多器官中诱导严重炎症并且可通过FADD/半胱天冬酶-8凋亡通路(REF)直接毒害细胞。进而已经在小鼠、非人灵长类动物和人类健康志愿者中测试了CBLB502并且发现其是短效炎症更温和的诱导剂。评估组织保护化合物时,也总有可能通过降低毒副作用,也可使肿瘤细胞抗性更强并且危及抗肿瘤治疗的功效。测试了皮下生长肿瘤和实验性肝转移的CT-26结肠癌小鼠模型中用CBLB502和抗Fas抗体联合治疗的体内抗肿瘤作用。在最近公开的研究中使用了这种肿瘤模型,该研究应用表达FasL的鼠伤寒沙门氏菌,总减毒细菌,将FasL递送至热带肿瘤并诱导Fas介导的抗肿瘤作用(Loeffler等2008)。通过RT-PCR和NF-kB依赖性萤光素酶报告基因测定确定,CT-26肿瘤细胞和A20淋巴瘤细胞不表达TLR5(图14)。此时,单独用抗Fas抗体或在单独注射抗Fas抗体24h和1h前给予2次重组CBLB502的组合处理荷瘤小鼠(4μg/只小鼠,图9D)。将处理小鼠体内的皮下生长肿瘤的体积与完整小鼠体内生长的肿瘤的体积作比较。发现CT-26肿瘤相当耐毒,但是不耐致死剂量的抗Fas抗体(图9D)。用CBLB502预处理稍微使肿瘤对抗Fas抗体敏感,在生长抑制肿瘤反应中反映出来。在通过脾内注射表达萤光素酶的CT-26肿瘤细胞,接着进行脾切除术诱导的肝转移的实验模型中测试了Fas介导的抗肿瘤作用。处理后每4-6天,使用Xenogen萤光素酶成像来评估肝肿瘤生长。在每个成像过程计数仍然没有肝肿瘤生长的小鼠(图9E)。结果证明通过两种处理,单独的抗Fas抗体或在用CBLB502预处理后给药,肝脏中的肿瘤出现(图9G)和生长显著延迟。TLR5阴性CT-26肿瘤对用抗Fas和CBLB502联合处理的敏感性增强表明了对CT-26肿瘤的抗肿瘤免疫应答的活化。实际上,对抗Fas/CBLB502处理24h后取得的具有CT-26肿瘤的肝脏样品的免疫组织化学分析揭示了嗜中性粒细胞在肿瘤结节内部和周围积聚(图9F)。因此,CBLB502并未保护肿瘤免受抗Fas抗体毒性并且甚至可稍微增强Fas介导的对CT-26肿瘤的抗肿瘤作用。同时保护正常肝组织免受Fas介导的毒性可使Fas激动剂的量增加,达到对肝转移的完全预防和对皮下生长肿瘤的治疗作用。
材料和方法
小鼠
从NCI(Frederick,MD)购买NIH-Swiss雌性小鼠,从Jackson Laboratory(BarHarbor,ME)购买BALB/c和C57B1/6雌性小鼠。在实验中使用的所有小鼠均为10-14周龄。最初从Xenogen(Alameda,CA)购买具有NF-kB诱导型萤光素酶报告基因的Balb/C-Tg(IκBα-luc)Xen小鼠并在我们家养的群体中繁殖。
试剂
从Cleveland BioLabs,Inc.获得细菌鞭毛蛋白衍生物CBLB502。从Sigma购买来自大肠杆菌055:B5的细菌脂多糖(LPS)。从BD Biosciences购买纯化激动性仓鼠抗小鼠Fas抗体,克隆Jo2。
使用NF-kB报告小鼠模型分析NF-κB活化
为BALB/c-Tg(IκBα-luc)Xen报告小鼠皮下注射CBLB502(0.2mg/kg)。处理2h后,通过非侵入性体内成像检测CBLB502对NF-kB的诱导(图1A)。对小鼠注射D-萤光素(3mg/100μl,腹腔内,Promega),立即用异氟烷麻醉并且用Xenogen IVIS成像系统100系列照相。为量化结果,注射2、6和24h后获得来自皮下注射了100μl PBS、CBLB502(0.2mg/kg)或LPS(1mg/kg)的NF-kB报告小鼠的肝脏、肺、肾脏、脾脏、心脏和肠道样品(图7B、10)。使含有蛋白酶抑制剂混合物的溶解缓冲液(根据生产商的建议,Calbiochem)覆盖组织样品以获得100mg组织/1ml溶解缓冲液。随后,在4C、14,000rpm下匀化并离心10min。添加30μl萤光素试剂(Bright-Glo Luciferase Assay System,Promega)后立即测量20μl样品中的萤光素酶活性。将萤光素酶活性标准化为每μg的蛋白质提取物。按TLR配体处理小鼠的肝脏和从注射PBS的对照小鼠获得的肝脏中平均萤光素酶单位之间的比例计算萤光素酶诱导倍数。
p65易位的免疫组织化学染色。
检测从皮下注射了CBLB502(0.04mg/kg)或LPS(1mg/kg)的NIH-Swiss小鼠分离的肝脏中的p65位置。向对照小鼠注射PBS。在处理20、40和60min后获得组织样品,将其处理成石蜡块。用兔多克隆抗体为所用肝脏组织染色NF-kB p65和兔单克隆抗体染色细胞角蛋白8,接着用适当的二次荧光染料偶联抗体染色(p65-绿色,细胞角蛋白-8-红色)。在板上用从NIH-Swiss小鼠输注了EGTA(0.5mM于PBS中)的肝脏组织分离的原代小鼠肝细胞和从(BDBiosciences)购买的人肝细胞培养物进行相同染色,接着对原代小鼠肝细胞进行胶原酶消化。在体外经指定时间用CBLB502(100ng/ml)或LPS(1μg/ml)处理两种类型的肝细胞。对照肝细胞保持完整。在×20放大倍数下照相(图7C、D、E)。
存活率测定
NIH-Swiss小鼠腹腔内注射2、3、4和5μg于200μl PBS中的抗Fas抗体以测定100%致死剂量,发现这个小鼠品系的100%致死剂量为4μg/只小鼠。然后在4μg抗Fas抗体(腹腔)前30min、2h和6h皮下注射CBLB502(0.04mg/kg,皮下)(图8A)。通常因抗Fas肝毒性的死亡在抗体注射后前1-2天内发生。观察并记录30天内的小鼠存活率。
肝脏中凋亡细胞的TUNEL染色
在石蜡包埋样本中检测在抗Fas抗体30min之前注射CBLB502(皮下,0.04mg/kg)或PBS5h后NIH-Swiss小鼠肝脏中的细胞凋亡。用TUNEL POD试剂盒(Roche AppliedScience),通过间接末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记(TUNEL)法将凋亡细胞染色(图8B)。
肝脏形态的组织学评估
在抗体前30min,用或不用CBLB502(0.04mg/kg)预处理,注射抗Fas抗体5h(图2C)或5、12和26h后动态地(图S3)从NIH-Swiss小鼠收集肝脏样本。使用未经处理(“完整”)的小鼠作为对照。将组织样本于10%缓冲福尔马林中固定,包埋于石蜡中,切片并且经苏木精伊红染色处理。
肝脏出血的组织学染色
用Cy5偶联的小鼠IgG的抗体为石蜡切片染色[Jackson Immunoresearch,伪紫色]并且用具有DAPI的ProLong Gold防褪色剂封固[Invitrogen,蓝核染色]。通过红色自体荧光使红细胞在红色通道中显现(图2D)。使用Axio Vision软件(rel.4.6.3),在装备有AxioCam HRc1300万像素数码相机的Axiolmager Z1荧光显微镜(Zeiss)下拍摄图像。
半胱天冬酶活化
将肝脏切为小片并用组织研磨器(Bullet Blender,NextAdvance)匀化于补充了2mM DTT的缓冲液(10mM Hepes、0.4mM EDTA、0.2%CHAPS、2%甘油)中。所有步骤均在冰上进行。在13,000xg下离心肝脏匀浆20min,并且将上清液储存在-20℃下。通过用50μM于200μl含10mM HEPES、0.4mM EDTA、0.2%CHAPS、2%甘油和2mM DTT的无细胞系统缓冲液中的荧光底物乙酰基-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-氨基甲基香豆素(Ac-DEVD-amc)(ENZO,LifeSciences)培养肝脏匀浆(含50μg总蛋白)来测定半胱天冬酶活性。在37℃下培育0和2h后,用荧光计(Ex:355,Em:485)(Victor3,PerkinElmer)测量荧光amc的释放。数据表示为twp和0h之间的差异(图8E)。
检查注射或未注射CBLB502,经抗Fas抗体处理的小鼠血清中的谷丙转氨酶(ALT)
在抗Fas抗体前30min对NIH-Swiss小鼠(3只/组)皮下注射1μgCBLB502。根据生产商的说明(Stanbio Laboratory,Boerne,TX,USA),使用商用酶测定法确定小鼠血清中存在谷丙转氨酶(ALT)。以60s间隔(△A/min)测量340nm下的吸光度(图8F)。
蛋白质印迹分析
使用补充了蛋白酶抑制剂混合物(Sigma-Aldrich St.Louis,MO)的RIPA缓冲液(Sigma-Aldrich St.Louis,MO),从经处理和未经处理的小鼠肝脏中分离出总蛋白。通过在变性4-20%聚丙烯酰胺Novex凝胶(Invitrogen,Carlsbad,CA)中电泳来分离蛋白提取物并将其转移到尼龙聚偏二氟乙烯(PVDF)膜(Immobilon-P,Millipore Billerica MA)上。使用下列抗体:半胱天冬酶-8抗体(Calbiochem,Darmstadt,Germany)、抗BID(AbCam,CambridgeMA)。从Santa Cruz Biotechnology,Inc.(Santa Cruz,CA)购买辣根过氧化物酶(HRP)偶联的二次抗兔和抗小鼠抗体(图9A和9B)。
RNA分析
使用TRIzol试剂,根据生产商说明(Invitrogen,Carlsbad,CA)从经处理和未经处理的小鼠肝脏萃取总RNA。为消除基因组DNA的任何最终污染,用DNA酶I(Invitrogen,Carlsbad,CA)处理分离的RNA。根据生产商说明,使用SuperScriptTM II逆转录酶和寡聚(dT)12-18引物(Invitrogen,Carlsbad,CA)来合成cDNA。通过RT-PCR检测用CBLB502和LPS处理30min和2h的完整小鼠的肝脏中Bcl2A1B、Bcl2A1D、IER-3、Fos、Jun和JunB基因的RNA表达。使用GAPDH作为对照以监测对基因表达的诱导。使用LaserGene软件(DNASTAR,Inc.,Madison,WI)设计引物,然后使用UCSC Genome Browser In-Silico PCR网站检查定位引物。使用对IER3基因(GenBank登记号NM_133662.2)(有义链5’-ACTCGCGCAACCATCTCCACAC-3’和反义链5’-CTCGCACCAGGTACCCATCCAT-3’)、Bcl2A1B基因(GenBank登记号NM_007534.3)(有义链5’-TAGGTGGGCAGCAGCAGTCA-3’和反义链5’-CTCCATTCCGCCGTATCCAT-3’)、Bcl2A1D基因(GenBank登记号NM_007536.2)(有义链5’-TCTAGGTGGGCAGCAGCAGTC-3’和反义链5’-ATTCCGCCGTATCCATTCTCC-3’)、Jun(GenBank登记号NM_010591.2)(有义链5’-TGAAGCCAAGGGTACACAAGAT-3’和反义链5’-GGACACCCAAACAAACAAACAT-3’)、Fos(GenBank登记号NM_010234.2)(有义链5’-GAGCGCAGAGCATCGGCAGAAG-3’和反义链5’-TTGAGAAGGGGCAGGGTGAAGG-3’)、JunB(GenBank登记号NM_008416.2)(有义链5’-AGCCCTGGCAGCCTGTCTCTAC-3’和反义链5’-GTGATCACGCCGTTGCTGTTGG-3’)和GAPDH基因(有义链5’-ACCACAGTCCATGCCATCAC-3’和反义链5’-TCCACCACCATGTTGCTGTA-3’)有特异性的引物。使用每个基因的特异性引物对,进行cDNA扩增20-30个循环(图3C)。
具有CT-26肿瘤的小鼠的实验治疗
用2个同源结肠腺癌CT-26肿瘤模型:1)CT-26皮下生长肿瘤和2)CT-26肿瘤的实验性肝转移模型,分析CBLB502对肿瘤对抗Fas抗体的敏感性的影响。用携带萤光素酶基因的慢病毒载体,在萤光素酶组成型表达的CMV启动子下转导CT-26细胞。通过在BALB/c小鼠的两腹侧皮下注射CT-26肿瘤细胞(2.5×105个/100μl)来诱导肿瘤。当肿瘤直径达到约4-5mm时,将小鼠随机分为3个组并开始处理。一组小鼠腹腔内注射抗Fas抗体(4μg/只小鼠),另一组在抗Fas抗体注射(4μg/只小鼠)前24h和1h,用CBLB502(1μg/只小鼠)处理。对照小鼠(“完整”)接受皮下和腹腔PBS注射代替CBLB502和抗体。每2天,使用卡尺测量并通过下式计算肿瘤体积:V=Π/6*a2*b,其中a<b。由于对照组有大肿瘤而终止实验时,跟踪存活率2周(图9D)。使用ANOVA单向方差分析评估肿瘤体积之间的统计差异(p<0.05)。为了肝脏肿瘤生长的发展,直接将CT-26肿瘤细胞(2×105个/50μl)注射至脾脏,接着在5min后进行脾切除术。用抗Fas抗体和CBLB502与抗体的组合,以对肿瘤细胞接种后第5天开始的皮下肿瘤描述的相同方式处理小鼠。在肿瘤细胞注射后第14、17、22和28天,使用Xenogen IVIS成像系统100系列对用异氟烷麻醉并注射了D-萤光素(3mg/100μl,腹腔内)的小鼠进行非侵入性生物发光成像。当确定肝脏中的肿瘤生长时处死小鼠。使用对数秩(Mantel-Cox)检验进行无肝脏肿瘤曲线的统计比较(p<0.05)(图9G)。
实施例2
无胸腺小鼠异种模型中CBLB502对结肠HCT116腺癌皮下生长的抗肿瘤活性。向8只无胸腺裸鼠的2个腹侧皮下注射HCT116(0.5×106个/100μl PBS)以诱导肿瘤。当肿瘤直径变成约3-5mm时(到注射后第6天),将小鼠随机分成2组,5只小鼠为CBLB502处理组而3只小鼠为PBS对照组。测定用CBLB502处理的小鼠体内对肿瘤生长的抑制。图15中示出了数据。
实施例3
无胸腺小鼠异种模型中CBLB502对293-TLR5皮下肿瘤生长的抗肿瘤活性。向10只无胸腺裸鼠的2个腹侧皮下注射肿瘤细胞(2×106个/100μl PBS)以诱导肿瘤。当肿瘤直径变成约3-5mm时(到注射后第7天),将小鼠随机分成2组,5只小鼠为CBLB502处理组而5只小鼠为PBS对照组。在用CBLB502处理的小鼠体内发现对肿瘤生长的抑制。图16中示出了数据。
实施例4
无胸腺小鼠异种模型中CBLB502对A549腺癌皮下生长的抗肿瘤活性。向8只无胸腺裸鼠(0.5×106个/100μl PBS)的2个腹侧皮下注射原始A549细胞(ATCC,CLL-185)以诱导肿瘤。当肿瘤直径变成约3-5mm时(到注射后第6天),将小鼠随机分成2组,5只小鼠为CBLB502处理组而3只小鼠为PBS对照组。对具有A549肿瘤的小鼠注射CBLB502(1μg/只小鼠)或PBS三次,时间间隔24h。在注射PBS的对照组小鼠中,肿瘤体积逐渐有规律地增长。另一方面,注射CBLB502的小鼠在注射后前几天表现出抑制肿瘤生长,然后肿瘤生长恢复。第一次处理2周后,在肿瘤生长重新开始之前,第二轮CBLB502注射(第14、15和16天)诱导类似肿瘤生长抑制约1-2周。因此,到实验结束时,2组小鼠体内A549肿瘤的尺寸显著不同,用CBLB502处理的小鼠比用PBS处理的小鼠小很多。图17中示出了数据。
实施例5
CBLB502对同源原位(皮下)生长的鳞状细胞癌SCCVII肿瘤的抗肿瘤作用。CBLB502或PBS(未处理)处理(0.1mg/kg,皮下,第1、2、3天)后,同源C3H小鼠中SCCVII原位肿瘤生长的速率达到肿瘤尺寸400mm3,n=6-10。图18中的x轴表示用和不用CBLB502处理时肿瘤达到400mm3体积所需的天数。图18中示出了数据。
实施例6
CBLB502在具有晚期Ward结直肠癌的Fischer大鼠体内的抗肿瘤活性。经5天腹腔内施用CBLB-502每天一次(0.2mg/kg×5次剂量),在肿瘤移植至4只大鼠体内5天后开始。4只对照大鼠接受作为媒介物对照的PBS注射。每天测量一次肿瘤重量。在用CBLB502处理的3只大鼠体内观察到完全反应(肿瘤完全消失)(图19)。该组中第4只大鼠的肿瘤生长与对照组的大鼠相似。
实施例7
CBLB502注射对TLR5表达不同的A549肿瘤(A549-shTLR5对比A549-shV)的作用。为了抑制TLR5表达,用表达对人TLR5基因有特异性的shRNA的慢病毒pLKO1-puro载体[CCG-GCC-TTG-CCT-ACA-ACA-AGA-TAA-ACT-CGA-GTT-TAT-CTT-GTT-GTA-GGC-AAG-GTT-TTT-G]或对照空载体(shV,Sigma-Aldrich,St.Louis,MO)转导在NF-kB启动子(Cellecta,MountainView,CA)控制下表达萤火虫萤光素酶基因的A549细胞。嘌呤霉素选择后,根据生产商方法(Promega,Cat#E4530,Madison,WI),使用萤光素酶报告测定来测试A549-shV和A549-shTLR5细胞响应于用CBLB502处理的NF-kB活化。然后,将A549-shV和A549-shTLR5细胞(1×106个/100μl PBS)皮下注射至20只无胸腺裸鼠的2个腹侧以诱导肿瘤。用CBLB502或作为对照的PBS处理具有皮下生长的A549-shV和A549-shTLR5肿瘤异种移植物的小鼠(5只小鼠/组)。结果证明,重复施用单独的CBLB502导致A549-shV(表达TLR5)肿瘤异种移植物中的肿瘤生长速率降低,这证明了药物的直接肿瘤抑制作用。对A549衍生肿瘤显示,这种作用依赖于TLR5,因为由慢病毒转导shRNA对人TLR5引起的TLR5敲除致使A549肿瘤不再对CBLB502的直接抗肿瘤作用敏感。图20中示出了数据。
实施例8
CBLB502注射对TLR5表达不同的H1299肿瘤(H1299-对照对比H1299-TLR5)的作用。为了诱导TLR5表达,用表达人TLR5基因的慢病毒构造来转导H1299细胞(原为TLR5阴性)。响应于CBLB502处理,通过IL-8生成来检查TLR5的功能活性。然后将两种肿瘤细胞类型(1×106个/100μl PBS)皮下注射至无胸腺裸鼠的2个腹侧以诱导肿瘤。与上述A549模型相似,用CBLB502或作为对照的PBS处理荷瘤小鼠。结果证明,重复施用单独的CBLB502仅仅导致H1299-TLR5(表达TLR5)肿瘤异种移植物中的肿瘤生长速率降低,这证明了药物的直接肿瘤抑制作用。对H1299(TLR5阴性)显示,肿瘤生长不受CBLB502处理影响。图21中示出了数据。
实施例9
该实施例证明,膀胱组织是CBLB502的强响应器。如以上对肝脏组织所述进行实验。在皮下注射100μl PBS、CBLB502(0.2mg/kg)或LPS(1mg/kg)2h后评估报告小鼠的肝脏、小肠(回肠部分)、结肠、脾脏、肾脏、肺和心脏中的NF-kB依赖性萤光素酶表达。检测了每一组3只小鼠体内每μg蛋白质提取物的标准化萤光素酶活性。图22中示出了数据。
实施例10
表2示出了小鼠靶器官中CBLB502转录活化基因的图谱(示出了膀胱结果)。注射1和3h后,根据其功能聚集在用CBLB502处理的小鼠的膀胱中强烈上调的基因。最大一类由趋化因子、细胞因子及其受体组成,这表明了先天免疫调动机制的活化。
实施例11
将不表达TLR5的CT-26肿瘤细胞皮下注射至同源BALB/c小鼠中以诱导肿瘤。用CBLB502(0.04mg/kg,皮下)处理荷瘤小鼠,间隔24h给药2次。将处理小鼠体内的皮下生长肿瘤的体积与完整小鼠体内生长的肿瘤的体积作比较。用CBLB502预处理对肿瘤生长无任何影响。然后在通过脾内注射表达萤光素酶的CT-26肿瘤细胞(图23B和C)和A20淋巴瘤细胞(图23D),接着进行脾切除术诱导的肝转移实验模型中测试了CT26肿瘤生长。处理后每4-6天,使用Xenogen萤光素酶成像来评估肝肿瘤生长。在每个成像过程计数仍然没有肝肿瘤生长的小鼠。结果证明,在两个肿瘤模型中通过CBLB502处理防止了肿瘤生长并显著延迟了肿瘤出现。肝肿瘤模型(B、C、D)中CBLB502处理组和对照组之间的差异显著(对数秩p<0.05)。图23中示出了数据。
实施例12
CBLB502对Fas介导的肝毒性的防护。A.单独腹腔注射4μg抗Fas抗体或在抗体前30min、2h和6h组合注射CBLB502(1μg/只小鼠)后NIH-Swiss小鼠的的存活率。括号内是每次处理小鼠的数量。B.保护肝脏免受抗Fas抗体毒性。使用TUNEL技术检测注射抗Fas抗体5h后肝脏内的细胞凋亡。经苏木精伊红染色的组织形态显示了注射抗Fas抗体对肝脏的坏死损害和CBLB502的保护。通过组织中的红细胞浸润、小鼠IgG对照(紫色)和DAPI细胞核(蓝色)来检测肝脏出血。图24中示出了数据。
实施例13
保护肝脏免受TNF-α和LPS毒性。A.注射TNF-a或LPS5h后检测半胱天冬酶3/7并且在注射24h后,在TPS/TNF-a前30min用和未用CBLB502处理的小鼠体内检测脂质氧化(表明炎症性损伤)。通过CBLB502注射防止肺中由TNF(1mg/只小鼠)诱导的半胱天冬酶活化和脂质氧化。通过在LPS前30min注射CBLB502完全消除了LPS(10mg/只小鼠)诱导的损伤效应。按处理24h后的蛋白质浓度标准化数据,n=3。如果在h-TNF前30min注射CBLB502,小鼠肝脏中无半胱天冬酶活化(TNF注射5h后)并且脂质氧化少得多(TNF注射24h后,如炎症性损伤所表明)。B.免疫组织化学分析(苏木精伊红染色)确认通过在TNF-a之前注射CBLB502保护了肝脏完整性。与完整对照相比,TNF处理小鼠的肝脏显示肝细胞的空泡形成在门静脉周稍微更明显并且依赖于剂量(在TNF0.4mg/只小鼠时更严重)。在用0.2mg或0.4mg/只小鼠的CBLB502和TNF处理的小鼠肝脏中,变化最小并且尽管仍可见轻微空泡形成,但是肝细胞接近正常。图25中示出了数据。
实施例14
保护肺免受TNF-a和LPS毒性。与完整对照相比,TNF处理小鼠的肺显示肺泡细胞反应性增生、充血、间质性水肿和肺泡渗出物,导致气腔减小,并且改变依赖于剂量(在TNF400时更严重)。在用200ng或400ng的CBLB502和TNF处理的小鼠的肺中,变化最小。尽管仍可见轻微的肺泡空泡增厚,但是形态接近正常(图26B)。如果在LPS(10mg/kg)或h-TNF(0.05mg/kg)前30min注射CBLB502,肺中的脂质氧化(表明炎症性损伤)的水平几乎正常(图26A)。图26中示出了数据。
实施例15
通过注射CBLB502保护小鼠免于口服致死鼠伤寒沙门氏菌。图27中示出了实验条件。
实施例16
该实施例证明伊立替康消除了鞭毛蛋白的抗肿瘤作用。图28中示出了数据。用CBLB502(0.2mg/kg)处理具有皮下生长的同源Ward结肠肿瘤的Fischer大鼠,经3天腹腔内施用CBLB502,每天一次。每次CBLB502注射30min后,静脉注射伊立替康(200mg/kg)。使用PBS作为媒介物对照(图28A)。CBLB502使大鼠免遭伊立替康毒性,伊立替康抗肿瘤活性不受干扰(图28B)。然而,在伊立替康处理大鼠中未观察到CBLB502的抗肿瘤作用(图28C)。这证明,CBLB502的抗肿瘤作用需要足够的先天免疫水平。
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Claims (7)
1.Toll样受体(TLR)激动剂在制备用于治疗哺乳动物中不表达TLR5的癌症的药物中的应用,其中所述癌症存在于表达TLR5的肝组织中,且所述TLR激动剂由SEQ ID NO:8的氨基酸序列组成。
2.TLR激动剂在制备减少哺乳动物的癌症复发的药物中的应用,其中所述癌症不表达TLR5且存在于表达TLR5的肝组织中,所述癌症复发选自转移和肿瘤再生,且所述TLR激动剂由SEQ ID NO:8的氨基酸序列组成。
3.根据权利要求1或2所述的应用,其中所述癌症为肿瘤或转移性的,其中所述癌症选自黑素瘤、结肠癌、乳腺癌、前列腺癌和恶性血液肿瘤,且其中所述恶性血液肿瘤为淋巴瘤。
4.根据权利要求1所述的应用,其中所述激动剂作为单一疗法施用和/或在切除肿瘤之前、之后或同时施用。
5.根据权利要求1所述的应用,其中所述哺乳动物未接受联合癌症治疗或化学治疗或放射治疗。
6.根据权利要求1所述的应用,其中所述癌症是结肠癌。
7.根据权利要求1的应用,其中所述癌症是淋巴瘤。
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