CN105263513A - 调控调节性t细胞功能的方法和组合物 - Google Patents
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Abstract
药物组合物包含一个从化合物号为1,2,3,4,5,6,7,13,22,23,24和25中选择出的化合物(如表1所述),和一个药学上可接受的赋形剂。本发明的药物组合物可进一步包含一个抗原,和/或一个佐剂。也提供了一种抑制调节性T细胞(Treg)介导免疫抑制的方法,或笼统地说通过使用含有一个能够激活MyD88-IRAK4信号通路的人Toll样受体(TLR)8配体的药物组合物,提供了一个增强免疫应答的方法。本发明进一步提供一种筛选Treg细胞抑制宿主免疫应答抑制剂的方法,其中所述Treg细胞表达CD25,GITR和FoxP3,分泌IL-10,能够抑制CD4+T细胞活性。
Description
发明领域
本发明涉及免疫学,具体涉及增强机体免疫反应的方法,更具体地说是涉及逆转调节性T细胞对机体免疫反应抑制的方法。
相关申请的交叉引用
本申请要求申请日为2013年3月14日、专利申请号为61/781,024的美国临时专利申请的优先权,该专利申请内容通过参考并入本申请中。
发明背景
通过调控免疫系统来消除传染物、恶性肿瘤细胞等是一个很有前景的治疗方法。直到最近这种治疗方法在肿瘤免疫上的应用也仅仅有零星的成功案例(DiLorenzoetal.,2011;Lesterhuisetal.,2011;Rosenberg,2011)。最近FDA批准通过了免疫治疗性疫苗/药物sipuleucel-T(Provenge)和ipilimumab(Yervoy)代表肿瘤免疫治疗领域里程碑式的成就(Hodietal.,2010;Kantoffetal.,2010)。此外,应用gp100肽治疗黑色素瘤的三期临床试验取得了很好的临床效果(Schwartzentruberetal.,2011)。然而,这些药物的临床效果远远没有达到完全有效和永久治愈的程度。对于sipuleucel-T,病人使用后也只延长4.1个月的存活期,没有明显的肿瘤消退及PSA水平的变化;而每个病人的花费却高达93,000美元。因此,急需要为癌症患者,包括转移性前列腺癌患者,开发一种更为有效和平价的疫苗/药物。
多种因素可造成肽为基础的疫苗,包括FDA批准的疫苗临床效果差的原因(Buonerbaetal.,2011),其中,强有力的负调控机制,包括肿瘤微环境中调节性T细胞(Treg)介导的免疫抑制,是改善肿瘤疫苗和药物疗效的主要障碍Curieletal.,2004;Wangetal.,2004;WangandWang,2007;Zou,2006)。例如,原先就存在于肿瘤病灶位置的CD4+调节性T细胞(Treg),可以潜在地抑制抗肿瘤免疫反应,从而成为肿瘤免疫治疗的主要障碍(Wangetal.,2004;Wangetal.,2005;WrzesinskiandRestifo,2005)。CD4+Treg介导的肿瘤免疫抑制在肿瘤动物模型和肿瘤患者中都有报道(BerendtandNorth,1980;Mukherjietal.,1989),在不同种类肿瘤的患者,包括肺癌,乳腺癌及卵巢癌患者,其CD4+CD25+Treg细胞在全部CD4+T细胞中所占的比例升高(Curieletal.,2004;Wooetal.,2001)。我们的研究进一步证明肿瘤病灶中存在抗原特异性CD4+Treg细胞,它们可以诱导抗原特异性和局部免疫耐受(Wangetal.,2004;Wangetal.,2005)。因此克服这些免疫抑制可能是成功开发更有效的肿瘤疫苗和药物的关键。一些研究者试图用CD25+抗体消除CD4+CD25+Treg细胞(Mahnkeetal.,2007;Morseetal.,2008;Powelletal.,2008;RechandVonderheide,2009),另有研究者用环磷酰胺去除Treg细胞(Audiaetal.,2007;Ghiringhellietal.,2007)。然而,无论是CD25抗体还是环磷酰胺均不是特异针对Treg细胞,因为CD25不仅仅表达在CD4+CD25+Treg,还表达在活化的T细胞表面。
Treg细胞根据其表型、细胞因子表达谱和抑制机制的不同可以分为不同的亚类。自然发生的CD4+CD25+Treg细胞是CD4+T细胞的一个小亚群,它发源于胸腺,未接触抗原,通过细胞接触机制抑制免疫反应(Sakaguchi,2004;Shevach,2002)。相比之下,抗原诱导的Treg细胞,例如Tr1和Tr3是在外周中经抗原刺激生成,它们通过释放抑制性的细胞因子IL-10/TGF-β来抑制免疫反应(Levingsetal.,2002;Shevach,2002;vonBoehmer,2005)。我们之前的研究表明LAGE1或ARTC1特异的CD4+Treg细胞与自然发生的CD4+CD25+Treg细胞一样,依赖于细胞接触机制抑制免疫反应(Wangetal.,2004;Wangetal.,2005)。CD4+CD25+Treg细胞和LAGE1特异的Treg细胞的抑制功能在DCs不存在的条件下可以被TLR8配体如多聚鸟苷核苷酸(PolyG-OND)逆转(Pengetal.,2005)。因为CD4+Treg细胞富集在肿瘤部位,我们推测,Treg细胞的不同亚类可能存在于肿瘤病人的肿瘤浸润性淋巴细胞(TILs)中。我们在研究验证这一推测时发现了一个新型抗原特异性CD4+Treg细胞亚群,它的免疫抑制作用是通过释放不同于IL-10或TGF-β(或二者都有)的可溶性因子。
尽管用PolyG-OND处理可以逆转Treg细胞功能(Kiniwaetal.,2007;Pengetal.,2005;Pengetal.,2007),但是还不确定PolyG-OND是否可以逆转人和小鼠Treg细胞的抑制功能。
急需一种方法和治疗试剂可用来操控Treg细胞的免疫抑制能力。
发明内容
这里描述的是产生一种新型的CD4+Treg细胞,它抑制免疫反应是通过IL-10或TGF-β非依赖性的可溶性因子介导的。为克服免疫抑制及去除免疫抑制细胞所引起的潜在问题,本发明利用新鉴定的Treg细胞开发了一种筛选系统用于筛选、鉴定能够阻断Treg细胞抑制功能的化合物。分别用新鉴定的化合物处理Treg细胞不同的时间,可以得到维持Treg细胞处于非抑制状态的不同时间窗口期。由于小鼠的TLR8没有功能,我们制备了人TLR8转基因小鼠,研究发现,用本发明抑制Treg细胞功能的化合物分别处理人TLR8转基因小鼠及C57BL/6野生型小鼠,人TLR8转基因小鼠而不是野生型小鼠的抗肿瘤免疫反应增强。
宿主免疫反应中的Treg细胞抑制剂可以增强机体免疫反应,可用于在病人的免疫系统需要增强的情况下,如针对肿瘤或感染性疾病时,使用。
抑制或增强宿主免疫反应中的Treg细胞功能的化合物、包含化合物的药物组合物包括但不局限于表1所列出的代表性化合物号为1,2,34,5,6,7,13,22,23,24和25的化合物。
因此,一个实施方案中,本发明提供了一个药物组合物,其中含有达到药学有效量的、来源于表1所列化合物号为1,2,34,5,6,7,13,22,23,24和25中的一个化合物,以及符合药学规范的赋形剂。本发明中的药物组合物可进一步包含一个抗原,这个抗原可能蛋白,多核苷酸或多糖抗原。在一个实施方案中,本发明的药物组合物还可进一步包含一个佐剂。
在另一个实施方案中,本发明在哺乳动物需要时提供了一种抑制Treg细胞介导的免疫抑制的方法,或更通俗地说是增强机体免疫反应的方法,此方法是给哺乳动物用一个有效药物剂量的、激活MyD88-IRAK4信号通路的一个Toll样受体8配体的药物组合物。所述配体选自ssRNA40,ssRNA33,CpG,Poly-G10,resiquimod,loxoribine,鞭毛蛋白,LPS,Pam3CSK4;或从化合物号为1,2,34,5,6,7,13,22,23,24和25的组中选取。
在一个实施方案中,哺乳动物是人。哺乳动物可能患有癌症或者是具有患癌症的风险。可进一步给予哺乳动物致免疫量的、含有癌症特异性抗原的癌症疫苗。在另一个实施方案中,进一步给予哺乳动物佐剂。佐剂可与抗原一起或偶联到抗原上给药。
在另一个实施方案中,有效剂量的化疗药物也可给予哺乳动物。
哺乳动能可能患感染性疾病或有得感染性疾病的风险。
本发明进一步提供了筛选抑制宿主免疫反应的Treg细胞抑制剂的方法,包括1)提供一个候选化合物,2)提供CD4+Treg细胞,3)分别在候选化合物存在及不存在的条件下,将初始型CD4+T细胞与CD4+Treg细胞共培养,4)分别在候选化合物存在及不存在的条件下,检测初始型CD4+T细胞的生长速率,5)比较在候选化合物存在与否的条件下CD4+T细胞的生长速率,如果化合物存在的条件下CD4+T细胞的生长速率比化合物不存在时要高,说明此化合物可以逆转CD4+Treg免疫抑制功能,可以作为CD4+Treg的抑制剂。CD4+Treg细胞表达CD25,GITR和FoxP3,释放IL-10,可以抑制机体的免疫反应。CD4+Treg细胞也可对某种抗原有特异性。在一个实施方案中,本发明的一种筛选方法包括将CD4+T细胞与抗原呈递细胞(APCs),如呈递特异抗原的DCs共培养。CD4+Treg细胞的生长速率可以通过掺入CD4+T细胞的[3H]-胸苷来检测。
提供本发明所述的化合物或者药物组合物,单独使用或联合其他治疗剂,例如抗原制剂或疫苗的方法治疗所需病人(例如对于癌症病人或患有感染性疾病的病人)。
附图简要说明
图1.肿瘤反应性CD4+CD25+T细胞系或克隆的产生和表征。(A)用流式细胞仪分析CD4+CD25+T细胞在患者初始型CD4+T细胞或其TIL108中的情况。(B)自TIL108来源的细胞中分析抗原特异性T细胞克隆。测试T细胞克隆对一系列肿瘤靶标及表达HLA-DR1,DR4或DR7分子的293T细胞的反应。586mel,1363mel,1558meland164mel表达HLA-DR1分子,而108meland1359mel分别为HLA-DR7和-DR4阳性。T细胞释放的GM-CSF通过ELISA检测。
图2.细胞因子表达谱及流式细胞仪分析CD4+T细胞的结果。(A)T细胞克隆的细胞因子表达谱。(B)FACS分析T细胞克隆。用藻红蛋白(PE)或FITC标记的单克隆抗体靶向CD4,CD25和GITR分子。同型对照抗体作为阴性对照。(C)实时定量PCR检测TIL108T细胞中Foxp3的表达水平。TIL1363-Th细胞作为对照。HPRP作为内参。
图3.TIL108Treg细胞抑制免疫反应的功能特征。(A)TIL108CD4+Treg克隆的免疫抑制活性。所有的TIL108Treg克隆抑制初始型CD4+T细胞的增值,而CD4+效应1363-Th细胞增强初始型CD4+T细胞的增值。(B)免疫抑制不依赖于细胞接触机制。通过transwell系统将初始型CD4+T和Treg细胞克隆分开培养。TIL108Treg克隆与初始型T细胞不直接接触时也存在对初始型T细胞增殖的抑制。(C)用IL-10抗体和TGF-β抗体不能逆转恢复初始型T细胞的增值。(D)对初始型T细胞的抑制是通过TIL108Treg细胞的培养上清介导的。10微升的Treg细胞克隆培养基足以抑制初始型T细胞的增值。(E)抑制初始型T细胞增殖所需培养上清的滴定。
图4.TIL108上清抑制CD8+和CD4+效应细胞的增殖和IL-2的分泌。(A)TIL108Treg细胞的培养上清抑制CD8+效应细胞的增值。从TIL108Treg克隆中分离的培养上清可以抑制CD8+效应细胞的增值,而CD4+T细胞和TIL1359细胞培养上清却不能。按照要求选用不同量的培养上清用于测试。(B)TIL108细胞的培养上清抑制CD4+效应细胞IL-2的释放。用OKT3抗体处理的TIL108Treg细胞培养上清可以抑制TIL1363CD4+效应细胞IL-2的产生。相比之下,未经处理的TIL108Treg细胞和经OKT3抗体处理的TIL1558-Th细胞的培养上清不能抑制TIL1363CD4+效应T细胞IL-2的分泌。(C)用OKT3抗体处理TIL108细胞比其细胞培养上清对TIL1363CD4+效应细胞IL-2的释放抑制作用更强。TIL108Treg细胞用OKT3或对照抗体预处理12小时,接着用T细胞培养基洗涤。TIL1558-Th细胞做对照。这些T细胞分别与TIL1363CD4+T细胞和1363mel靶细胞混合培养。
图5.处理TIL108Treg细胞后,可逆转其培养上清的免疫抑制作用。(A)通过Poly-G10寡核苷酸逆转TIL108Treg功能,检测细胞培养上清的免疫抑制作用。(B)经Poly-G10寡核苷酸处理的TIL108Treg细胞培养上清不能抑制初始型T细胞的增值。未经处理的Treg细胞培养上清作为对照。(C)用特异的siRNA敲低Treg细胞中TLR8,MyD88及IRAK4的表达,可阻断Treg细胞抑制初始型T细胞增殖的可逆性。TLR7和TLR9的siRNA作为对照。(D)TIL108Treg细胞的免疫抑制功能可以被天然合成的人TLR8配体抑制,但不能被其他TLR配体抑制。(E)用合成的或天然的人TLR8配体而非其他的TLRs的配体处理TIL108Treg细胞,细胞培养上清可以恢复初始型T细胞的增值。
图6.在小鼠Treg细胞存在的条件下,TLR配体恢复初始型T细胞增殖的能力。(A)自然发生的CD4+CD25+Treg细胞用CD4和CD25抗体染色,通过流式细胞仪分离和纯化。用初始型T细胞分别与不同数量的CD4+CD25+Treg细胞反应,用以测定Treg细胞的免疫抑制活性。初始型T细胞(1x105)与纯化的APC(1x104)与Treg细胞在含有0.5μgCD3抗体的培养基中共孵育。共培养56小时后,加入胸苷(终浓度为1μCi/孔),继续培养16小时。用液体闪烁计数器测定胸苷的掺入量。实验一式三份。Treg或APC细胞单独不会对CD3抗体应答。(B)在TLR配体存在与否的条件下,初始型T细胞与Treg按1∶0.5的比例检测T细胞/APC的增值。检测方法与(A)部分相似。Pam3CSK4poly(I:C)、LPS、鞭毛蛋白、loxoribine和resiquimod、poly-G10、CpG分别是TLR2、TLR3、TLR4、TLR5、TLR7、TLR8、TLR9的配体.每个配体的工作浓度以商家说明及我们的滴定实验为准。
图7.TLR8在转基因小鼠中的表达,功能及抗肿瘤免疫。(A)人TLR8基因在CD4-hTLR8转基因小鼠中的表达。从小鼠各个组织和各种类型的细胞中提取总RNA,用于RT-PCR检测hTLR8的表达。(B)功能评价表达人TLR8的Treg细胞对Poly-G3OND的应答反应。
图8.Poly-G3阻断Treg细胞功能增强抗肿瘤免疫。CD4-hTLR8Treg小鼠在第-2,-1天分别用Poly-G3或Poly-T10(对照)OND(1.0μg/小鼠)处理,第0天皮下接种4种不同类型的肿瘤细胞。每两天检测肿瘤的大小。
图9.两种筛查方法用于鉴定能够阻断Treg细胞免疫抑制作用的小分子化合物。
图10.鉴定抑制Treg细胞功能的小分子化合物。这些小分子化合物包含一个喹诺酮结构。那些对初始型CD4+T细胞的增值有显著影响的(CPM>60K,与单独初始型T细胞相比恢复50%)可作为药物开发的良好选择。不被任何化合物处理的,单独的幼稚T细胞或CD4+幼稚T细胞+Treg细胞作为对照。
图11.三种不同的化合物分别处理Treg细胞不同的时间后,检测Treg细胞功能逆转的持久性。用三种不同的药物预处理Treg细胞1天,3天和8天,之后用不含药物的培养基培养直至检测其免疫抑制功能。
发明描述
CD4+调节性T(Treg)细胞因可以抑制机体对自身组织及肿瘤细胞的免疫反应而引起自身免疫耐受,但是对Treg细胞不同亚群引发的免疫抑制机制还知之甚少。本发明发现一种具有新的免疫抑制机制的抗原特异性CD4+Treg细胞亚群。这种新的抗原特异的CD4+Treg细胞亚群与其他Treg细胞一样表达CD25,GITR以及FoxP3标志物,释放IL-10,但是它并不是通过IL-10及TGF-β抑制免疫反应,而是通过可溶性因子。研究发现,用人的Toll样受体(TLR)8的配体处理Treg细胞可以逆转其免疫抑制,TLR8可以活化MyD88-IRAK4通路,提示TLR-MyD88信号通路调控引发免疫抑制的可溶性分子的释放可能是一个调控免疫抑制的新机制。
因此,本发明提供了一种可以逆转CD4+调节性T(Treg)细胞抑制机体免疫反应的方法。本发明还提供了免疫刺激组合物以及治疗/预防的方法,包括给予受试者本发明中的免疫刺激的组合物。本发明还提供了可以逆转CD4+调节性T(Treg)细胞对机体免疫抑制的小分子化合物的筛选方法。本发明提供的方法和组合物可以提高某些免疫治疗的免疫效果,尤其是在用肿瘤特异抗原疫苗治疗或预防肿瘤方面。
药物应用本发明的Treg细胞抑制剂可用于预防和治疗多种炎性疾病,哮喘,特应性皮炎,荨麻疹,过敏性疾病(过敏性支气管肺曲霉病,过敏性嗜酸性肠胃炎及类似病症),肾炎,肾病,肝炎,关节炎,慢性类风湿关节炎,牛皮癣,鼻炎,结膜炎,缺血再灌注损伤,多发性硬化症,溃疡性结肠炎,急性呼吸窘迫综合症,细菌感染性休克,糖尿病,自身免疫病,移植排斥,免疫抑制,癌症转移,获得性免疫缺陷综合症等等。
在一些实施方案中,本发明的药物组合物还包括一个抗原。当施加的适量抗原被呈递,与组合物中的其他组分联合,可以有效的产生针对此抗原的免疫反应。可以产生免疫反应的抗原的有效剂量可以通过现代技术手段很容易确定。
抗原可以与药物组合物中的任意成分同时或依次使用。因此,该抗原可以单独使用,或与一种或多种刺激产生免疫反应的佐剂(包括本发明中的调节性T细胞抑制剂)同时使用。在一些实施方案中,抗原可以与某种佐剂同时使用(例如以混合物形式),但是随后再依次使用一个或多个佐剂。
抗原与药物组合物中的其他成分连续共同施用时,抗原至少和组合物中的一种共同施用,这样尽管抗原和药物组合物中的其他成分没有同时施用,它们可以在给药部位同时存在。抗原与免疫刺激组合物中的成分顺序施用时也存在以下情况:抗原或药物组合物中至少一种成分已经在治疗部位代谢完,但是,在后续药物组合物中的一种或多种成分在治疗部位应用前,至少抗原或先前使用的其他成分中的一种在治疗部位还存在细胞效应(例如细胞因子的释放,某种细胞的活化等)。
抗原可以是能够诱导TH1免疫反应的任何物质,可以引起以下一种或多种反应,例如能够引起CD8+T细胞反应,NKT细胞反应,γ/σT细胞反应或TH1抗体反应。合适的抗原包括,但不局限于肽,多肽,脂类,糖脂,多糖,碳水化合物,多核苷酸,朊病毒,灭活或未灭活的细菌,病毒或真菌,以及细菌,病毒,真菌,原生动物,肿瘤来源或生物衍生抗原,毒素或类毒素。此外,目前的某些实验抗原,尤其是重组蛋白,糖蛋白以及肽等不能诱导很强免疫应答,可以与本发明提供的Treg细胞的抑制剂联合使用。示范性实验亚基抗原包括与以下病毒疾病相关的抗原:如腺病毒疾病,艾滋病,水痘,巨细胞病毒疾病,登革热,猫白血病病毒感染症病毒感染症,鸡瘟,甲肝,乙肝,HSV-1,HSV-2,猪瘟,甲型流感,乙型流感,日本脑炎,麻疹,副流感,狂犬病,呼吸道合胞病毒疾病,轮状病毒疾病,疣和黄热病。在某些实施方案中,抗原可以是癌抗原或肿瘤抗原。在这里癌抗原和肿瘤抗原的专业名词可以互换使用,均指癌细胞差异高表达的抗原。
本发明的药物组合物可用于细胞介导的免疫反应可治疗的相关疾病。这样的组合可至少包含一个有效治疗剂量的Treg细胞抑制剂,可进一步包含一个有效剂量的抗原。
在治疗方案中,此药物组合物可以作为单一治疗使用,或与另一种治疗剂,如抗病毒剂,抗生素等联合使用。
由于可以增强免疫力,本发明提供的药物组合物特别适用于,但不局限于治疗以下疾病:(a)病毒性疾病,如腺病毒,疱疹病毒引起的疾病(例如HSV-I,HSV-II,CMV或VZV),痘病毒引起的疾病(例如天花,牛痘或传染性软疣),小核糖酸病毒引起的疾病(例如鼻病毒或肠病毒),正粘病毒(例如流感病毒)引起的疾病,副粘病毒(例如猪副流感病毒,腮腺炎病毒,麻疹病毒和呼吸道合胞病毒(RSV))引起的疾病,冠状病毒引起的疾病(例如SARS),乳多空病毒(例如,乳头瘤病毒,比如那些导致生殖器疣,寻常疣,或跖疣)引起的疾病,肝DNA病毒(例如乙型肝炎病毒)引起的疾病,黄病毒(例如C型肝炎或登革热病毒)引起的疾病或逆转录病毒(例如慢病毒如HIV);(b)细菌性疾病,例如因感染细菌而引发的疾病,这类细菌包括:埃希氏菌属,肠杆菌属,沙门氏菌属,葡萄球菌属,志贺氏菌,李斯特菌,气杆菌,螺杆菌,克雷伯氏菌,变形杆菌,假单胞菌属,链球菌属,衣原体,支原体,肺炎球菌细菌感染引起的疾病,奈瑟氏菌属,梭菌属,芽孢杆菌属,棒状杆菌属,分枝杆菌属,弯曲杆菌属,弧菌属,沙雷氏菌属,普罗维登斯菌属,色杆菌,布鲁氏菌,耶尔森氏菌属,嗜血杆菌属,或博代氏杆菌;(C)其他传染性疾病,例如衣原体,真菌引起的疾病,例如念珠菌病,曲霉病,组织胞浆菌病,隐球菌性脑膜炎,或寄生虫病(疟疾及其他),卡氏肺孢子虫肺炎,利什曼病,隐孢子虫病,弓形体病和锥虫感染;(D)肿瘤性疾病,例如上皮内瘤变,宫颈不典型增生,光化性角化病,基底细胞癌,鳞状细胞癌,肾细胞癌,卡波西氏肉瘤,黑色素瘤,肾细胞癌,白血病,(骨髓性白血病及其他),慢性淋巴细胞性白血病,多发性骨髓瘤,非何杰金氏淋巴瘤,皮肤T细胞淋巴瘤,B细胞淋巴瘤,和毛细胞白血病,以及其他癌症(例如,上述确定的癌症);(E)TH2介导遗传性过敏症及自身免疫病,例如特应性皮炎或湿疹,嗜酸细胞增多,哮喘,过敏症,过敏性鼻炎,系统性红斑狼疮,原发性血小板增多症,多发性硬化症,Ommen综合征,盘状狼疮,斑秃,抑制瘢痕瘤形成和其他类型的疤痕,伤口愈合的增强,包括慢性伤口。
本发明的药物组合物在某些实施方案中可以用作佐剂,与能够增强体液/细胞介导的免疫反应的物质联合使用,例如肝病毒,细菌或寄生虫抗原;灭活的病毒,肿瘤来源的抗原,原生动物,组织来源抗原,真菌或细菌抗原,类毒素,毒素;自身抗原;多糖;蛋白质;糖蛋白;肽;细胞疫苗;DNA疫苗;重组蛋白;糖蛋白等等,在以下方面使用:例如,BCG,霍乱,鼠疫,伤寒,甲型肝炎,乙型肝炎,丙型肝炎,甲型流感,乙型流感,副流感,脊髓灰质炎,狂犬病,麻疹,腮腺炎,风疹,黄热病,破伤风,白喉,嗜血杆菌乙型流感,结核,脑膜炎球菌和肺炎球菌疫苗,腺病毒,艾滋病毒,水痘,巨细胞病毒,登革热,猫白血病病毒感染症,鸡瘟,HSV-1和HSV-2,猪瘟,日本脑炎,呼吸道合胞病毒,轮状病毒,乳头状瘤病毒,黄热病,和阿尔茨海默氏病。本发明的免疫刺激组合物也对自身免疫力低下患者有帮助。例如,可用于治疗机会性感染及由于抑制细胞介导的免疫力而引发的肿瘤,如移植患者,肿瘤病人及HIV病人。
基于上述目的,本发明的药物可以全身或局部给药,通常口服或肠胃外给药,可与其他任选药物组合。给药量取决于以下因素:例如年龄,体重,症状,所需治疗效果,给药方式以及治疗持续时间。对于成年人,给药量从1ng到1000mg一般通过口服给药,数天一次,3天一次,2天一次,1天一次或者一天数次不等,从1ng到100mg通过肠胃外给药(优选静脉内给药),数天一次,3天一次,2天一次,1天一次,甚至一天数次,或一天24小时持续静脉给药不等。如上文所述,本发明药物的用量随各种条件和临床状况改变。因此,也存在试剂用量低于或高于上述范围的情况,本发明的抑制剂可以以多种形式施用,例如固体形式口服,液体形式口服或以肠胃外给药的形式,如注射,外用药物,栓剂,滴眼剂,吸入剂等等,或与其他药物组合施用。
在一个实施方案中,本发明提供了一种筛选Treg细胞抑制剂的方法,包括1)提供候选化合物或测试剂,2)提供CD4+Treg细胞抑制免疫反应;3)在候选化合物存在及不存在的条件下,初始型CD4+T细胞与抑制免疫反应的CD4+Treg细胞共培养,可随机加入抗原呈递细胞,例如树突细胞,4)检测CD4+T细胞的生长速率,(例如[3H]-胸苷参入量)以及在候选化合物存在与不存在的条件下比较CD4+T细胞的生长速率,当候选化合物存在时,CD4+T细胞生长速率较快则说明候选化合物可以抑制Treg细胞对机体的免疫抑制。在一个实施方案中,CD4+Treg细胞是通过IL-10或TGF-β非依赖性的可溶性因子介导的免疫抑制反应机制。
如果药剂或要测试的化合物有显著的抑制效果,例如与未添加化合物的培养基(例如,自由培养基或其他对照)相比[3H]胸腺嘧啶的掺入量有至少20%的增加,则可认为其为Treg细胞活性的抑制剂。以上提到的方法可以用于筛选化合物库,例如那些基于合成化学或天然存在的化合物及其衍生物,一个例子就是MixtureBasedPositional筛选数据库,它是系统地将成千上万的化合物进行系统编号后提供相关信息。位置扫描技术已被成功应用于识别新的酶抑制剂,受体激活剂和拮抗剂,抗微生物,抗真菌,抗细菌的化合物(Houghtenetal.,J.Med.Chem.42:3743-3778,1999;Pinillaetal.,Nat.Med.9:118-122,2003)。此外,此项技术已被多个研究机构独立验证。有约50个实验室进行了至少100次独立研究(Houghtenetal.,J.Med.Chem.42:3743-3778,1999),证实系统整理筛选化合物的实用性,例如位置扫描数据库。
本文以下实例仅为示范性说明,本领域的技术人员在遵循本申请的精神和所附属的权利范围的条件下可进行改变。
示例
材料和方法
T细胞系和细胞克隆。CD4+肿瘤浸润性淋巴细胞(TIL108)是从一个黑色素瘤患者的新鲜肿瘤组织分离培养获得。所有的TILs和T细胞克隆用含10%人AB型血清及重组IL-2(300IU/ml)的RPMI640培养基培养。如上所述,T细胞克隆通过TIL108有限稀释法培养获得(平均0.3个细胞/孔)(Wangetal.,2002)。为了使细胞以最佳的状态扩增,如前所述,加OKT3扩增(Wangetal.,2002)。TIL1363-Th和TIL1558-ThCD4+细胞克隆分别从TIL1363和TIL1558分离培养获得,并分别与前述细胞相同或相似(Wangetal.,2004)。它们的名称是为了区别它们与其他CD4+Treg细胞的性质:即它们可以释放Th1细胞因子以及响应CD3抗体具有增强初始型CD4+T细胞增值的能力。T细胞释放的细胞因子的检测按上文所述进行(Wangetal.,2004)。
流式细胞仪分析。T细胞与GITR抗体孵育(R&D系统),随后用连接有FITC的山羊抗小鼠二抗染色,检测GITR的表达。细胞在流式细胞仪检测之前至少在含有300IU/ml的IL-2中培养2周。用连接有PE或FITC的CD4,CD25及GITR抗体(BDBiosciences)染色,分别检测T细胞CD4,CD25及GITR的表达。洗涤后,用流式细胞仪扫描分析。
增值测定。CD3单克隆抗体包被(2μg/ml)的96孔板中,初始型CD4T细胞(1x105)与调节性T细胞以不同的比例(1∶0.2,1∶0.1and1∶0.05)共培养。可以将TIL108或其他效应细胞(TIL1363-Th及TIL1558-Th)的培养上清加入到新鲜的培养基中,使总体积为200μl用于增值检测。培养56小时后,增殖的初始型及效应T细胞用[3H]胸苷标记(终浓度为1μCi/孔),培养16小时。用液体闪烁计数器测定[3H]胸苷掺入量。TIL108Treg细胞,在某些情况下,用Poly-G10,OKT3抗体或不同的TLR配体处理12小时,之后用PBS或T细胞培养基洗涤。在新鲜的培养基中培养24-36小时,收集这些T细胞的培养上清,检测其抑制活性。下面的配体购自Invivogene公司(SanDiego,CA):LPS(100ng/ml),CpG-A(3μg/ml),CpG-B(3μg/ml),imiquimod(10μg/ml),loxoribine(500μM),poly(I:C)(25μg/ml),ssRNA40/LyoVec(3μg/ml),ssRNA33/LyoVec(3μg/ml),pam3CSK4(200ng/ml),鞭毛蛋白(10μg/ml),或Poly-G3(3μg/ml)。Transwell实验按上文所述操作(Wangetal.,2004)。
实时定量PCR分析。用Trizol法提取T细胞(1x107)总RNA(Invitrogen,Inc.SanDiego,CA)。用SuperScriptIIRT试剂盒(Invitrogen,Inc.SanDiego,CA)逆转录。逆转录混合物含2μg总RNA,总体积为20μL,在42℃孵育1小时。Foxp3的mRNA水平通过实时定量PCRABI/PRISM7000系统检测(PEAppliedBiosystems,Inc.FosterCity,CA)。PCR用引物及Foxp3或HPRT特定的内部荧光TaqMan探针从PE应用生物系统公司购买(FosterCity,CA)。通过实时定量PCR检测每个样品TLR7,8,9,MyD88及IRAK4的表达,用HPRT的相对量为基准,评测上述基因表达情况,方法如上文所述(Pengetal.,2005)。
慢病毒为基础的siRNAs的构建及病毒转导。用电脑辅助程序为每个基因选择几个siRNA序列(19个核苷酸)。寡核苷酸包含一个siRNA序列,8个核苷酸间隔件,以及一个聚胸腺嘧啶终止子序列,退火后连接到表达GFP的pLentilox3.7载体上,双酶切位点为HapI和xhoI(Rubinsonetal.,2003)。IRAK4,MyD88,及TLR7,,8和9的siRNAs的病毒转导如上文所述(Pengetal.,2005)。转染3天或4天后通过流式细胞仪的ARIA分拣系统区分GFP+及GFP-细胞,检测转染效率。分离到的Treg细胞在功能性增值检测系统中通过Poly-G10的检测其可逆性。
结果和讨论
CD4+TIL细胞系的免疫抑制活性的鉴定
为构建肿瘤特异性CD4+T细胞系,我们先用肿瘤患者的手术切除的新鲜肿瘤样本培养肿瘤浸润性淋巴细胞。去除CD8+T细胞后,检测CD4+T细胞对一系列肿瘤细胞系的反应。TIL108是一个肿瘤反应CD4+T细胞系,被选中进一步研究。流式分析表明,TIL108在总的CD4+T细胞群中含有17%CD4+CD25+T细胞,而正常PBMC来源的CD4+T细胞群体含有约6%CD25+T细胞(图1A)。为检测这些细胞的功能,我们发现,用CD3抗体分离初始型CD4+T细胞,TIL108细胞可以抑制其增值反应(图1A)。而对照组的CD4+T细胞的增值反应没有被抑制,说明CD4+TIL108细胞系中富集抗原特异的CD4+Treg细胞克隆。
接下来,我们采用一种有限稀释法从TIL108细胞株获得了CD4+T细胞克隆。35种肿瘤反应性CD4+T细胞克隆中,有12种克隆被成功扩增来获得大量T细胞用于进一步分析。正如图1B所示,所有12种TIL108克隆能够特异性识别MHCII类匹配的108种黑色素细胞,其中3种识别异体1558黑色素瘤细胞,然而它们都不会对MHCII类匹配的EBV和293衍生的细胞系发生反应。
分泌IL-10的CD4+Treg细胞克隆的表型分析
为了确定每种T细胞克隆表达的细胞因子谱,我们发现所有12种T细胞克隆分泌大量GM-CSF,IFN-γ和IL-10,但很少或根本没有其它细胞因子(图2A)。通过FACS分析,我们展示了所有分泌IL-10的T细胞克隆表达CD4,CD25和GITR标志物。4种克隆的代表性数据如图2B所示。实时PCR分析揭示叉头状转录因子的表达在分泌IL-10的CD4+T细胞克隆中比在CD4+TIL1363辅助性细胞中高(图2C)。这些数据合起来表明TIL108T细胞克隆表达标志物和细胞因子通常与CD4+调节性T细胞有关。Treg细胞分泌可溶性因子介导CD4+T细胞的免疫抑制。
符合上述表型特征,所有的TIL108Treg细胞克隆对比TIL1363辅助性效应细胞,以剂量依赖的方式强烈抑制抗CD3诱导的效应CD4+T细胞增殖,TIL1363辅助性效应细胞增强而不是抑制初始型CD4+T细胞增殖(图3A)。为了确定这些TIL108Treg细胞是如何抑制初始型T细胞增殖,我们进行了侵袭实验。正如图3B所示,孔里的TIL108Treg细胞克隆仍然能够抑制孔外的初始型CD4+T细胞的增殖,此过程不受孔内对照T细胞的影响。因此,Treg细胞克隆的抑制功能不需要细胞-细胞接触。
我们接下来研究了Treg细胞克隆产生的大量IL-10是否参与免疫抑制。在试验培养基中添加抗10,抗10R或两者都有(每种10μg)不能恢复初始型CD4+T细胞的增殖活性(图3C)。添加大量抗体(30μg)或使用抗体包被的培养板(数据未展示)也获得了类似结果。第三种抗体,抗TGF-β,也不会对初始型CD4+T细胞的增殖活性产生任何影响(数据未展示)。这些结果表明IL-10和TGF-β不参与由TIL108Treg细胞克隆介导抑制初始型T细胞增殖的过程中。
为了直接测试细胞培养上清液是否能够抑制初始型CD4+T细胞的增殖,我们发现在功能性试验培养基中仅仅添加10μl来自TIL108Treg细胞克隆的细胞培养上清液,就能抑制90%以上的初始型CD4+T细胞增殖,然而添加相同量来自TIL1363辅助性细胞或初始型CD4+T细胞培养上清液增强而不是抑制初始型CD4+T细胞增殖(图3D)。滴定实验表明抑制活性随上清液量增加而增加(图3E)。这些结果表明TIL108Treg细胞分泌的可溶性因子与其抑制功能直接相关。因此,TIL108Treg细胞系/克隆也许代表了一类新的Treg细胞亚群,这群细胞介导免疫抑制通过一种不同于自然发生的CD4+CD25+Treg或Tr1/Th3细胞产生的免疫抑制机制(Levingsetal.,2002;Sakaguchi,2004;Shevach,2002)。随着评估大量肿瘤患者来源的临床样品的增加,很可能新型Treg亚群将被发现。除了CD4+Treg细胞,其他Treg细胞亚群,包括CD8+Treg细胞,NKT和γδTCRT细胞也可能在调节不同疾病类型(自体免疫疾病和癌症)的宿主免疫反应中起重要作用(Cortesinietal.,2001;HaydayandTigelaar,2003;JiangandChess,2004),表明存在一系列具有不同的抑制机制或表型的Treg亚群。
由TIL108Treg细胞抑制效应T细胞功能
我们接着测试了Treg细胞上清液是否能够抑制抗原特异性CD8+效应细胞的增殖。如图4A所示,上清液也可以强烈抑制CD8+TIL1359T细胞的增殖。为了确定由TIL108Treg细胞克隆分泌的可溶性因子是否能够抑制TIL1363辅助性效应细胞分泌IL-2的活性,我们用TIL1363辅助性细胞与1363黑色素瘤细胞(刺激物)在150μl新鲜培养基中共培养,添加50μl来自TIL108T细胞克隆的细胞上清液,加或不加抗CD3(OKT3)抗体刺激。来自TIL108Treg细胞的培养上清液用OKT3刺激抑制TIL1363-C1细胞分泌IL-2,对比来自无任何活性的OKT3刺激的TIL1558效应细胞培养上清液或来自不加OKT3刺激的TIL108Treg细胞培养上清液,。为了进一步检测TIL108Treg细胞与TIL1363辅助性T细胞和1363黑色素瘤细胞共培养是否能更好抑制IL-2,我们发现OKT3刺激的TIL108Treg细胞强烈抑制TIL1363辅助性T细胞分泌的IL-2对比不加OKT3刺激的TIL108Treg细胞(图4C)。TIL1558辅助性T细胞用OKT3抗体刺激不能抑制TIL1363辅助性T细胞分泌IL-2。这些数据表明抑制CD4+辅助性T细胞分泌IL-2需要激活TIL108Treg细胞。
逆转TIL108Treg细胞的抑制功能
我们接下来测试TIL108Treg细胞的抑制功能是否可以被Poly-G寡核苷酸抑制。将TIL108Treg细胞用Poly-G10预处理可导致TIL108Treg细胞的抑制功能被逆转且初始型CD4+T细胞增殖能力恢复,然而不处理TIL108Treg细胞,其抑制功能仍然存在(图5A)。更重要的是,从Poly-G10处理的TIL108Treg细胞收集的上清液增强初始型CD4+T细胞的增殖能力,对比来自未处理的TIL108Treg细胞,这些细胞仍然保留抑制能力(图5B)。表明由TIL108Treg细胞分泌的可溶性因子可被Poly-G介导的信号通路直接控制。
我们接下来寻求确定Poly-G寡核苷酸逆转TIL108Treg细胞功能是否需要TLR8信号通路。因此,我们用表达GFP的siRNA慢病毒载体敲除了存在于TIL108Treg细胞的TLR8信号通路的一些关键分子如TLR8,MyD88和IRAK4,表达GFP的siRNA慢病毒载体能够抑制相应基因的表达(Pengetal.,2005)。TIL108Treg细胞转染靶基因特异性的siRNA病毒粒子被分为GFP+(转染)和GFP-(未转染)细胞并测试它们对Poly-G10寡核苷酸的反应。正如图5C所示,用siRNA特异性敲除TLR8,MyD88和IRAK4破坏Poly-G10寡核苷酸逆转TIL108Treg细胞的功能。相比之下,当TIL108Treg细胞转染TLR7和TLR9特异性的siRNA病毒时,抑制活性和可逆性都不会受到影响。筛选的GFP-TIL108Treg细胞具有与未转染的亲本细胞相同的可逆转的抑制功能(图5C)。与这个结果一致,两个人TLR8的天然配体(ssRNA40和sssRNA33)也能逆转TIL108Treg细胞的抑制功能,然而TLRs的其他配体在Treg细胞存在时,不能恢复初始型型CD4+T细胞的增殖(图5D)。尽管ssRNA33配体效果欠佳,但是用Poly-G2和ssRNA40处理的TIL108Treg细胞上清液也获得了类似结果(图5D)。
由于像自然发生的CD4+CD25+Treg细胞和LAGE1-特异性CD4+Treg细胞一样,TIL108Treg细胞分泌的可溶性因子的抑制活性也直接受TLR8信号控制。不管抑制的具体机制,我们推测TLR8-MyD88信号通路特异性控制负责Treg细胞抑制作用的分子功能。尽管还不清楚为什么是TLR8而不是其他TLR信号通路,控制Treg细胞的抑制活性,Treg细胞中TLR的表达模式可以部分解释TLR8信号通路的独特性质。
另外一种情况是Treg细胞中的TLR8-MyD88-IRAK4复合物在MyD88-IRAK4的下游,可能招募一种独特的信号通路,这种信号通路被用于控制Treg细胞的功能。
通过独特的TLR配体调节小鼠Treg细胞功能
由于小鼠TLR8没有功能(Jurketal.,2002),逆转小鼠的Treg细胞抑制可能与人的Treg细胞抑制不同。为了试验Poly-G寡核苷酸是否能小鼠TLR8逆转Treg细胞的功能,我们分离并纯化了小鼠CD4+CD25+Treg细胞,评价它们对TLR配体的应答能力。小鼠Treg细胞能够抑制初始型CD4+T细胞的增殖,而单独的Treg细胞或APCs细胞加入抗CD3抗体后不会增殖(图6A)。然而,Poly-G寡核苷酸对逆转小鼠Treg细胞的抑制活性不起作用(图6B)。
有趣的是,TLR2,TLR7和TLR9的TLR配体能够逆转小鼠Treg细胞对初始型T细胞增殖的抑制作用(图6B)。LPS,一种TLR4配体,对逆转Treg细胞的抑制作用有轻微作用,然而TLR3和TLR5的配体对抑制功能毫无作用。这些数据确定特异性TLR信号在小鼠Treg细胞存在下控制初始型T细胞增殖,阐述了通过改变CD4+Treg和T辅助细胞的平衡来增强抗肿瘤免疫的基本原理。
构建人TLR8转基因模型
由于小鼠TLR8不起作用,我们最近生产了转基因(Tg)小鼠。人TLR8在脾脏,胸腺,淋巴结和CD4+T细胞中,但是在B细胞,CD8+T细胞或其他被分析的组织中不表达(图7A)。因此,在CD4人TLR8转基因小鼠的CD4+T细胞的人TLR8准确可靠表达及其功能将大大方便我们控制体内的Treg细胞功能。用Poly-G3而不是Poly-T10(对照组)处理表达人TLR8的Treg细胞,可以逆转它们的抑制功能(图7B)。这些研究表明通过Poly-G3可以逆转表达人TLR8的小鼠Treg细胞的抑制功能。
通过阻断Treg细胞功能增强抗肿瘤免疫
为了测试Poly-G3处理是否可以通过阻断Treg细胞功能增强体内抗肿瘤免疫,我们在day-2和day-1将Poly-G3,Poly-T10或CpG寡核苷酸通过尾静脉注入C57BL/6野生型(对照组)和CD4-人TLR8转基因小鼠体内,开发了一种保护性肿瘤模型。这种经过处理的小鼠在day0皮下注射B16肿瘤细胞(1×105细胞/小鼠),每2天监测肿瘤生长。我们发现Poly-G3处理增强CD4-人TLR8转基因小鼠而不是C57BL/6小鼠的抗B16肿瘤免疫反应(图8)。Poly-T10和CpG处理不能抑制C57BL/6小鼠和CD4-人TLR8转基因小鼠的肿瘤生长,表明Poly-G3诱导的抗肿瘤免疫需要CD4+T细胞表达TLR8。重要的是,我们发现Poly-G3处理抑制CD4-人TLR8转基因小鼠的前列腺RM1肿瘤细胞生长(图8)。在一些其它肿瘤类型包括淋巴瘤,MCA28肉瘤,和RM1前列腺癌肿瘤细胞中也获得了类似结果。
鉴定能够逆转人Treg细胞抑制功能的新化合物
由于目前我们对TLR8信号通路的认识还很有限,一种替代方法将是筛选小分子化合物。为了解释这种方法的可行性,我们已经从Timtec股份有限公司购买数百个小分子化合物,将如图9描述进行试验中筛选。
尤其是,用可溶性抗CD3抗体和抗原提呈细胞(APCs)进行了功能筛选试验。用磁珠(MiltenviBiotec)纯化PBMCs得到初始型CD4+T细胞。DCs用PBMC中的单核细胞在IL-4(500ng/ml)和GM-CSF(800ng/ml)条件下培养7天。1x105的初始型CD4+T细胞与调节性T细胞在包含2x104DCs和抗CD3抗体(100ng/ml)的200μl的培养基中以1∶0.2,1∶0.1和1∶0.05不同比例共培养,该培养基同时包含多种小分子化合物(1μM)。经过56h的培养,向每孔加入[3H]胸苷,终浓度为1μCi/孔,接着培养16h。[3H]胸苷的插入通过液体闪烁计数器测量。所有实验一式三份。此外,用抗CD3抗体不加APCs进行筛选:1x105初始型CD4T细胞与调节性T细胞在抗CD3单克隆抗体包被(2μg/ml)的96孔板中加或不加多种小分子化合物(1μM),以1∶0.2,1∶0.1和1∶0.05不同比例共培养。经过56h的培养,向每孔加入[3H]胸苷,终浓度为1μCi/孔,接着培养16h。[3H]胸苷的插入通过液体闪烁计数器测量。
我们鉴定出11个化合物可有效逆转Treg细胞的抑制功能(图10和表1)。这些结果表明小分子化合物能够逆转人Treg细胞的抑制功能。
药物诱导阻断Treg细胞抑制功能的持久性取决于药物的处理时间
为了确定药物诱导逆转Treg细胞抑制功能的持久性,我们在药物(低浓度)存在条件下分别培养Treg细胞(每份1x106)1,3,5天。Treg细胞不加药物培养作为对照组。Treg细胞在第1,3,8天收集并用PBS清洗3次除去残余药物。一些细胞(0.5-1x106)被用于功能检测,而剩余T细胞在包含IL-2的培养基中培养,且每隔4天被用于功能检测直至满3周。如图11所示,用化合物#1,#4和#7药物处理Treg细胞1天然后清洗后培养在不含药物的培养基中直至被用于检测。经过预处理的Treg细胞丧失抑制功能并维持没有抑制功能的状态5-6天。用药物处理Treg细胞3天后,其维持没有抑制功能的状态3-4周,然后再次恢复抑制功能。类似的,Treg细胞接受预处理8天,丧失抑制功能然后恢复抑制功能(图11)。因此,最有潜力的化合物,是由恢复至少50%的初始型T细胞增殖(例如阻断50%Treg细胞的抑制活性)所需的最低药物浓度以及维持没有抑制功能的状态来获得足够的时间诱导效应T细胞产生免疫反应的能力来确定,这对于进一步实验非常有用。由于药物诱导阻断Treg细胞抑制功能的持久性取决于药物的处理时间,我们可以在自体免疫反应风险最小时,控制时间窗来诱导最大抗肿瘤免疫。我们也希望鉴定小分子化合物增强Treg细胞的抑制功能。总之,我们的研究表明Treg细胞抑制功能肯以通过不同化合物/药物调节,这可能对于治疗许多类型的疾病非常重要。
表1化合物列表
本专利引用的所有出版文献,专利和专利申请通过参考并入本申请中适用于所有目的。在不偏离本发明的范围的情况下,任何具体实施方案的一个或多个特征可和任何其他具体实施方案中的一个或多个特征合并。上述描述是说明性的,而不是限制性的。本发明的许多变动对于本领域技术人员是显而易见。因此,本发明的范围不应根据上面的说明决定,而应根据所附权利要求及等同权利要求的全部范围决定。
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Claims (21)
1.一种药物组合物,包含药学有效量的化合物和药学上可接受的赋形剂,所述化合物选自化合物号为1,2,3,4,5,6,7,13,22,23,24或25。
2.权利要求1的药物组合物,进一步包含抗原。
3.权利要求1的药物组合物,进一步包含佐剂。
4.权利要求1的药物组合物,其中所述抗原为肽抗原,蛋白抗原,多核苷酸抗原,或多糖抗原。
5.在有需要的哺乳动物中抑制调节性T(Treg)细胞介导的免疫抑制或增强免疫应答的方法,所述方法包括给予哺乳动物药学有效量的、包含能激活MyD88-IRAK4信号通路的人Toll样受体(TLR)8配体的药物组合物。
6.权利要求5的方法,其中所述配体选自ssRNA40,ssRNA33,CpG,Poly-G10,瑞喹莫德,洛索立宾,鞭毛蛋白,LPS,Pam3CSK4。
7.权利要求5的方法,其中所述配体选自化合物号为1,2,3,4,5,6,7,13,22,23,24或25。
8.权利要求5的方法,其中所述哺乳动物为人。
9.根据权利要求5的方法,其中所述哺乳动物患有癌症或具有患癌症的风险,并且进一步给予所述哺乳动物致免疫量的、含有癌症特异性抗原的癌症疫苗。
10.根据权利要求9的方法,进一步给予哺乳动物佐剂。
11.根据权利要求10的方法,其中所述的癌症佐剂与抗原一起给予或偶联到抗原上。
12.权利要求11的方法,进一步包含给予有效量的化疗药物。
13.权利要求12的方法,进一步包含给予有效量的化疗药物。
14.根据权利要求5的方法,其中所述哺乳动物感染了传染病或处于患传染病风险。
15.筛选对Treg细胞的宿主免疫反应抑制性活性的抑制剂的方法,包括1)提供候选化合物,2)提供CD4+Treg细胞,3)在候选化合物存在或不存在的条件下,将初始型CD4+T细胞与CD4+Treg细胞共培养,4)在候选化合物存在或不存在的条件下,检测初始型CD4+T细胞的生长速率,以及5)比较在候选化合物存在与否的条件下CD4+T细胞的生长速率,其中化合物存在的条件下CD4+T细胞的生长速率比化合物不存在时更高,说明此化合物可以逆转CD4+Treg细胞的免疫抑制,并选择作为所述抑制剂。
16.根据权利要求15的方法,其中所述CD4+Treg细胞表达CD25,GITR和FoxP3;分泌IL-10,并能够抑制宿主免疫应答。
17.根据权利要求15的方法,其中所述CD4+Treg细胞具有抗原特异性。
18.根据权利要求17的方法,其中所述CD4+T细胞进一步与抗原提呈细胞(APCs)共培养。
19.根据权利要求18的方法,其中所述APCs是用于呈递抗原。
20.根据权利要求19的方法,其中所述APCs是树突状细胞。
21.根据权利要求15的方法,其中所述生长速率由[3H]-胸苷掺入CD4+T细胞的速率决定。
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WO2019160915A1 (en) | 2018-02-14 | 2019-08-22 | Dana-Farber Cancer Institute, Inc. | Irak degraders and uses thereof |
US11945800B1 (en) | 2023-09-21 | 2024-04-02 | King Faisal University | 1-cyclopropyl-6-fluoro-4-oxo-7-(4-((5-oxo-2-phenyl-4-(quinolin-2-ylmethylene)-4,5-dihydro-1H-imidazol-1-yl)methyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid as an anti-inflammatory and anticancer compound |
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