CN105473136A - 用于在有此需要的受试者中抑制淋巴细胞增殖的方法和药物组合物 - Google Patents
用于在有此需要的受试者中抑制淋巴细胞增殖的方法和药物组合物 Download PDFInfo
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- CN105473136A CN105473136A CN201480031935.4A CN201480031935A CN105473136A CN 105473136 A CN105473136 A CN 105473136A CN 201480031935 A CN201480031935 A CN 201480031935A CN 105473136 A CN105473136 A CN 105473136A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
Abstract
本发明涉及用于在有此需要的受试者中抑制淋巴细胞增殖的方法和药学组合物。具体的,本发明涉及CTP合酶1(CTPS1)抑制剂在用于在有此需要的受试者中抑制淋巴细胞增殖的方法中的用途。本发明也涉及用于筛选多个有用于在有此需要的受试者中抑制淋巴细胞增殖的测试物质的方法,包含由以下项组成的步骤i)测试每种测试物质其抑制CTPS1活性或表达的能力,和ii)鉴定抑制CTPS1活性或表达的物质从而鉴定有用于在有次需要的受试者中抑制淋巴细胞增殖的测试物质。
Description
发明领域
本发明涉及用于在有此需要的受试者中抑制淋巴细胞增殖的方法和组合物。
发明背景
淋巴细胞增殖是针对抗原(例如,病原抗原)的免疫反应的正常组成部分。然而在特定条件下,淋巴细胞增殖似乎是有害的。例如,器官移植引起复杂的系列免疫过程,其一般被归类为炎症、免疫、损伤组织的组织修复和结构强化。典型地,T细胞增殖通过分泌促炎症细胞因子,例如,白介素-2(IL-2)和IFN-g导致炎症。因此,本领域的技术人员已经尝试开发免疫抑制剂。免疫抑制药物分为五组:(i)基因表达调节因子;(ii)烷化剂;(iii)嘌呤从头合成的抑制剂;(iv)嘧啶从头合成的抑制剂;和(v)激酶和磷酸酶的抑制剂。例如,糖皮质激素主要通过抑制IL-2和其它介导物基因表达发挥免疫抑制和抗炎症活性。氨甲蝶呤和其聚谷氨酸衍生物通过释放腺苷抑制炎症性反应。霉酚酸和咪唑立宾(mizoribine)抑制肌苷单磷酸脱氢酶。霉酚酸诱导活化的T淋巴细胞的凋亡。环孢霉素和FK-506/他克莫司(Tacrolimus)抑制钙调神经磷酸酶(calcineurin)的磷酸酶活性。雷帕霉素抑制来自IL-2,表皮生长因子和其它细胞因子受体的信号转导。开发中的免疫抑制和抗炎症化合物包括p38激酶和环AMP磷酸二酯酶IV型同等型(其在T细胞中表达)的抑制剂。然而,免疫抑制剂由于它们的非特异性免疫抑制效果与毒性相关。减少免疫抑制能够防止与过度免疫抑制相关的副作用。然而,由于对于每对供体受体对,固有免疫抑制的要求未知,免疫抑制最小化带有免疫抑制不足和随之而来的急性排斥,移植物的过早丢失和死亡的潜在风险。免疫抑制药物一种有前景的应用是搜索通过新的机制抑制淋巴细胞增殖的试剂,因为目前使用的试剂都拥有非特异性的广泛免疫抑制效果。
发明简述
本发明涉及用于在有此需要的受试者中抑制淋巴细胞增殖的方法和组合物。
发明详述
由抗原识别和共信号触发的淋巴细胞功能意味着快速和强烈的细胞分裂,因此代谢适应1。胞苷核苷酸三磷酸(CTP)是一种DNA,RNA和磷脂代谢所需的前体2-4。CTP源自两种来源:补救途径和从头合成途径,所述从头合成途径依赖于两种酶,CTP合酶(或合成酶)1和2(CTPS1和CTPS2),虽然它们各自的功能未知5-7。CTP合酶活性是淋巴细胞中对于DNA合成潜在的重要步骤8,9。这里,发明人报道了对人类CTPS1中缺失功能突变(rs145092287)的鉴定,其引起新的,威胁生命的免疫缺陷,其特征在于活化的T和B细胞增殖能力的受损。在CTPS1缺陷受试者T和B细胞或使用shRNA敲低CTPS1的正常T细胞中,响应抗原受体介导的活化的增殖是缺陷性的。相反,近端和远端TCR信号传导事件和响应仅受到CTPS1缺乏的微弱影响。通过表达野生型CTPS1或通过添加外源性CTP或其核苷前体,胞苷,在CTPS1缺陷细胞中恢复了正常的T细胞增殖。在静息T细胞中发现CTPS1表达低,但在TCR活化后快速上调。这些结果突出了CTPS1通过免疫应答期间其维持活化的淋巴细胞增殖的能力,在免疫系统中关键且特异的作用。因此,CTPS1可能代表了能够特异性地抑制淋巴细胞活化的免疫抑制药物的治疗靶标。
因而,本发明的第一个方面涉及用于在有此需要的受试者中降低或抑制淋巴细胞增殖的方法,包含使用治疗有效量的至少一种CTP合酶1(CTPS1)抑制剂施用给所述受试者。
在一些实施方案中,本发明的方法适合用于抑制或减少T细胞增殖。
在一些实施方案中,本发明的方法适合用于抑制或减少B细胞增殖。
在一些实施方案中,所述受试者是移植的受试者。典型地,所述受试者可能已经使用选自下组的移植物移植:心脏,肾脏,肺,肝,胰腺,胰岛,脑组织,胃,大肠,小肠,角膜,皮肤,气管,骨,骨髓,肌肉,或膀胱。本发明的方法确实特别适合用于防止或抑制与接受体受试者对供体组织、细胞、移植物,或器官移植的排斥相关的免疫反应。移植相关疾病或病症包括移植物抗宿主病(GVDH),例如与骨髓移植相关,以及器官、组织,或细胞移植物移植(例如,组织或细胞同种异体移植或异种移植)排斥导致的或与其相关的免疫病症,包括,例如,皮肤,肌肉,神经元,胰岛,器官,肝的实质细胞移植物,等等。对于接受体受试者中的供体组织,细胞,移植物或实体器官移植物,据信根据本发明的CTPS1抑制剂可以有效的防止所述接受体中对这些移植物的急性排斥和/或用于长期维持治疗以防止所述接受体中这些移植物的排斥(例如,抑制患有糖尿病的受试者接受体中对来自供体的产生胰岛素的胰岛细胞移植物的排斥)。因此,本发明的方法对于防止宿主抗移植物病(HVGD)和移植物抗宿主病(GVHD)是有用的。所述CTPS1抑制剂可以在移植之前和/或移植后施用到所述受试者(例如,移植前至少一天,移植后一至五天,等等)。在一些实施方案中,所述CTPS1抑制剂可以在移植之前和/或移植后定期施用到所述受试者。
在一些实施方案中,所述受试者患有自身免疫疾病。如本文所使用的,“自身免疫疾病”是起因于并针对个体的自身组织的疾病或病症。自身免疫疾病的例子包括但不限于,阿狄森氏病(Addison'sDisease),过敏,斑秃(AlopeciaAreata),阿尔兹海默病,抗中性粒细胞胞浆抗体(ANCA)相关性血管炎,强直性脊柱炎,抗磷脂综合征(休斯综合症(HughesSyndrome)),关节炎,哮喘,动脉粥样硬化,动脉粥样硬化斑块,自身免疫性疾病(例如,狼疮,RA,MS,格雷夫斯病(Graves'disease)等),自身免疫性溶血性贫血,自身免疫性肝炎,自身免疫性内耳病,自身免疫性淋巴增生综合征,自身免疫性心肌炎,自身免疫性卵巢炎,自身免疫性睾丸炎,无精子症,贝切特氏病(Behcet'sDisease),贝格尔氏病(Berger'sDisease),大疱性类天疱疮(BullousPemphigoid),心肌病,心血管疾病,乳糜泻/腹腔疾病(CeliacSprue/Coeliacdisease),慢性疲劳免疫功能障碍综合征(CFIDS),慢性特发性多发性神经炎,慢性炎性脱髓鞘性,多发性神经根性神经病(Polyradicalneuropathy)(CIPD),慢性复发性多发性神经病(格林-巴利综合征(Guillain-Barrésyndrome)),丘-斯综合征(Churg-StraussSyndrome,CSS),瘢痕性类天疱疮,冷凝集素疾病(ColdAgglutininDisease)(CAD),慢性阻塞性肺病(COPD),CREST综合征,克罗恩氏病(Crohn'sdisease),皮炎,疱疹样皮炎(Herpetiformus),皮肌炎,糖尿病,盘状狼疮(DiscoidLupus),湿疹,获得性大疱性表皮松解症(Epidermolysisbullosaacquisita),原发性混合型冷球蛋白血症(EssentialMixedCryoglobulinemia),埃文氏综合征(Evan'sSyndrome),眼球突出(Exopthalmos),纤维肌痛,肺出血肾炎综合征(Goodpasture'sSyndrome),桥本氏甲状腺炎(Hashimoto'sThyroiditis),特发性肺纤维化,特发性血小板减少性紫癜(ITP),IgA肾病,免疫增生性疾病或病症(例如,银屑病),炎性肠病(IBD),包括克罗恩病和溃疡性结肠炎,胰岛素依赖型糖尿病(IDDM),间质性肺病,幼年型糖尿病,幼年型关节炎,幼年特发性关节炎(JIA),川崎氏病(Kawasaki'sDisease),兰伯特-伊顿肌无力综合征(Lambert-EatonMyasthenicSyndrome),扁平苔藓,狼疮,狼疮性肾炎,淋巴细胞性垂体炎(LymphoscyticLypophisitis),梅尼埃病(Ménière'sDisease),米勒费希尔综合症(MillerFisherSyndrome)/急性播散脑脊髓脊神经根病,混合型结缔组织病,多发性硬化症(MS),肌肉风湿,肌痛性脑脊髓炎(ME),重症肌无力,眼部炎症,落叶型天疱疮(PemphigusFoliaceus),寻常型天疱疮(PemphigusVulgaris),恶性贫血,结节性多动脉炎,多软骨炎,多腺体综合征(惠特克氏综合征(Whitaker'ssyndrome)),风湿性多肌痛,多发性肌炎,原发性无丙种球蛋白血症,原发性胆汁性肝硬化/自身免疫性胆管病(Autoimmunecholangiopathy),银屑病,银屑病关节炎,雷诺氏现象(Raynaud'sPhenomenon),莱特尔氏综合征(Reiter'sSyndrome)/反应性关节炎,再狭窄(Restenosis),风湿热,风湿性疾病,类风湿关节炎,结节病,施密特综合征(Schmidt'ssyndrome),硬皮病,综合症,僵人综合症(Stiff-ManSyndrome),系统性红斑狼疮(SLE),系统性硬皮病,大动脉炎,颞动脉炎/巨细胞动脉炎,甲状腺炎,1型糖尿病,2型糖尿病,溃疡性结肠炎,葡萄膜炎,血管炎,白癜风和韦格纳氏肉芽肿(Wegener'sGranulomatosis)。
如本文所使用的术语“CTPS1”具有其在本领域的一般意义并指代CTP合酶1。CTPS1是一种67-kDa的蛋白,含有CTP合成酶结构域和谷氨酰胺酰胺转移结构域,其从UTP和谷氨酰胺代谢形成CTP(Kursula,P.等,StructureofthesynthetasedomainofhumanCTPsynthetase,atargetforanticancertherapy.ActaCrystallogrSectFStructBiolCrystCommun62,613-7(2006))。
如本文所使用的,术语“CTPS1抑制剂”指代任意的天然或非天然化合物,其具有减少或抑制CTPS1的活性或表达的能力。典型地所述CTPS抑制剂能够通过结合所述蛋白直接作用于活性,或通过减少或抑制所述酶的表达间接作用于活性。因此CTPS1抑制剂包含CTPS1表达的抑制剂。例如,CTPS1抑制剂也包括能够与CTPS1的底物(即CTP或谷氨酰胺)竞争对应的催化结构域的任意化合物。典型的,所述抑制剂是小的有机分子或生物分子(肽,脂质,适体)。
在一些实施方案中,所述CTPS1抑制剂是氨基酸谷氨酰胺的任意功能性类似物,衍生物,取代产物,异构体,或同源物,其保留了谷氨酰胺结合CTPS1抑制剂的特性。
术语“谷氨酰胺类似物”在本文中意图包含上文提到的任意一个。根据本发明的谷氨酰胺类似物的制备通过本领域的技术人员公知的传统方法制备,见例如以下具体实施方案的上下文或标准参考文献中提到的参考文献。
在一些实施方案中,所述CTPS1抑制剂是正亮氨酸衍生物,例如6-重氮-5-氧-L-正亮氨酸(DON)。DON是一种谷氨酰胺类似物,其抑制许多各种不同的需要谷氨酰胺的反应,虽然主要的效果似乎在于哺乳动物细胞中的嘌呤的从头合成和CTP合成酶(Lyons,S.D.,Sant,M.E.,Christopherson,R.I.(1990)J.Biol.Chem.265,11377-11381)。其阻碍增殖并已经进行了作为癌症药物的广泛的临床试验(综述于Catane,R.,VonHoff,D.D.,Glaubiger,D.和L.Muggia,F.M.(1979)CancerTreat.Rep.63,1033-1038;和Ahluwalia,G.S.,Grem,J.L.,Hao,Z.,和Cooney,D.A.(1990)Pharmacol.Ther.46,243-271)。美国专利号2965634涉及正亮氨酸衍生物,例如DON,以及其生产的方法。
在一些实施方案中,所述CTPS1抑制剂是阿西维辛(acivicin)。阿西维辛已描述于美国专利号5,489,562。
在一些实施方案中,所述CTPS1抑制剂是UTP的类似物。这种类似物的例子是去氮尿苷(CAS号23205-42-7)。
其它的例子包括环戊烯基胞嘧啶(CPEC),吉西他滨(Gemcitabine)(2',2'-二氟脱氧胞苷,dFdC),放线菌素D,环己酰亚胺,联丁酰基环AMP,和6-氮杂尿苷。
“表达抑制剂”指代天然或合成的化合物,其具有抑制基因表达的生物效应。
在一些实施方案中,所述基因表达抑制剂是siRNA,反义寡核苷酸或核酶。
用于本发明中的基因表达抑制剂可以基于反义寡核苷酸构建物。反义寡核苷酸,包括反义RNA分子和反义DNA分子,将通过结合目标mRNA,作用以直接阻断目标mRNA的翻译,并从而防止蛋白翻译或增加mRNA降解,从而降低目标蛋白(即CTPS1)在细胞中的水平,以及活性。例如,可以合成至少约15个碱基且与编码目标蛋白的mRNA转录序列的特定区域互补的反义寡核苷酸,例如,通过常规的磷酸二酯技术和通过,例如静脉内注射或输注施用。使用反义技术的方法特异性抑制其序列已知的基因的基因表达是本领域公知的(例如,见美国专利号6,566,135;6,566,131;6,365,354;6,410,323;6,107,091;6,046,321;和5,981,732)。
小抑制性RNAs(siRNA)也可以充当基因表达的抑制剂用于本发明。可以通过使肿瘤,受试者或细胞与小双链RNA(dsRNA),或导致小双链RNA产生的载体或构建物接触,使得基因的表达被特异性的抑制(即RNA干扰或RNAi),降低基因表达。用于对其序列已知的基因选择适合的dsRNA或编码dsRNA的载体的方法是本领域公知的(例如,见Tuschi,T.等,(1999);Elbashir,S.M.等,(2001);Hannon,GJ.(2002);McManus,MT.等,(2002);Brummelkamp,TR.等,(2002);美国专利号6,573,099和6,506,559;和国际专利公开号WO01/36646,WO99/32619,和WO01/68836)。
核酶也可以充当基因表达的抑制剂用于本发明。核酶是酶促RNA分子,其能够催化RNA的特异性剪切。合酶作用的机制涉及核酶分子和互补靶标RNA的序列特异性杂交,随后核酸内切性剪切。因此,特异性并高效地催化目标mRNA序列的核酸内切性剪切的工程化发夹或锤头基序核酶分子在本发明的范围内有用。首先通过扫描目标分子的核酶剪切位点鉴定任意潜在RNA靶标内的特异性核酶剪切位点,其通常包括以下序列,GUA,GUU和GUC。一旦鉴定,对应含有剪切位点的目标基因区域的约15至20个核糖核苷酸之间的短RNA序列可以评价预测的结构特征例如二级结构,其可能使得所述寡核苷酸序列不适合。也可以通过使用,例如核糖核酸酶保护试验,测试其与互补寡核苷酸杂交的可及性(accessibility)来评价候选靶标的适合性。
反义寡核苷酸和核酶两者作为有用的基因表达抑制剂可以通过已知方法制备。这些方法包括用于化学合成的技术,例如,通过固相亚磷酰胺化学合成。或者,可以通过体外或体内转录编码RNA分子的DNA序列生成反义RNA分子。这种DNA序列可以整合到各种各样的载体中,所述载体整合了适合的RNA聚合酶启动子例如T7或SP6聚合酶启动子。可以引入对本发明的寡核苷酸的各种修饰,作为增加细胞内稳定性和半衰期的方法。可能的修饰包括但不限于,向所述分子的5’和/或3’端添加核糖核苷酸或脱氧核糖核苷酸侧翼序列,或在寡核苷酸骨架内使用硫代磷酸酯或2’-O-甲基而不是磷酸二酯酶连接。
本发明的反义寡核苷酸siRNA和核酶可以体内单独递送或与载体联合。在其最广泛的意义上,“载体”是能够促进反义寡核苷酸siRNA或核酶核酸转移到细胞的任意媒介物。典型地,所述载体将核酸转运到细胞,相对于所述载体不存在时将产生的降解程度具有减少的降解。一般来说,在本发明中有用的载体包括但不限于,质粒,噬菌粒,病毒,其它衍生于病毒或细菌来源的媒介物,其已经通过插入或整合所述反义寡核苷酸siRNA或核酶核酸序列操作。病毒载体是一种特别的载体类型,并包括但不限于来自以下病毒的核酸序列:逆转录病毒,例如莫洛尼鼠白血病病毒(moloneymurineleukemiavirus),哈维鼠肉瘤病毒(harveymurinesarcomavirus),鼠乳腺肿瘤病毒(murinemammarytumorvirus),和劳斯肉瘤病毒(rousesarcomavirus);腺病毒,腺相关病毒;SV-40型病毒;多瘤病毒;Epstein-Barr病毒;乳头瘤病毒;疱疹病毒;牛痘病毒;脊髓灰质炎病毒;和RNA病毒例如逆转录病毒。人们可以容易的采用未列出但本领域已知的其它载体。
具体的病毒载体基于非细胞病变性真核细胞病毒,其中非必须基因已经被感兴趣的基因取代。非细胞病变性病毒包括逆转录病毒(例如,慢病毒),其生命周期包含基因组病毒RNA逆转录为DNA随后原病毒整合到宿主细胞DNA中。逆转录病毒已经被批准用于人基因治疗试验。大多数有用的是复制缺陷的那些逆转录病毒(例如,能够指导所需蛋白的合成,但不能制造感染性颗粒)。这种遗传改变的逆转录表达载体对于基因在体内的高效率转导具有通用性。用于产生复制缺陷的逆转录病毒的标准方案(包括以下步骤:将外源性遗传材料整合到质粒中,使用质粒转染包装细胞系,由所述包装细胞系产生重组逆转录病毒,从组织培养基收集病毒颗粒,以及使用病毒颗粒感染目标细胞)提供于KRIEGLER(ALaboratoryManual,"W.H.FreemanC.O.,NewYork,1990)和MURRY("MethodsinMolecularBiology,"vol.7,HumanaPress,Inc.,Cliffton,N.J.,1991)中。
用于特定应用的具体病毒是腺病毒和腺相关病毒,其为双链DNA病毒,已经被批准在基因治疗中用于人类用途。所述腺相关病毒可以经工程化而是复制缺陷的,并能够感染许多各种不同的细胞类型和种类。其还具有优势例如,热和脂质溶剂稳定性;在不同品系的细胞中高转导频率,包括造血细胞;以及缺乏超感染抑制(superinfectioninhibition)从而允许多重系列转导。据报道,腺相关病毒可以以位点特异性的方式整合到人细胞DNA中,从而最大限度的减少插入诱变的可能性和逆转录病毒感染的插入基因表达特征的变化。另外,在不存在选择压力下,已经在组织培养中跟踪了野生型腺相关病毒感染大于100代,这意味着腺相关病毒基因组整合是相对稳定的事件。腺相关病毒也可以以染色体外的方式发挥作用。
其它载体包括质粒载体。质粒载体在本领域中已经广泛地描述且对于本领域的技术人员是公知的。见,例如SANBROOK等,"MolecularCloning:ALaboratoryManual,"SecondEdition,ColdSpringHarborLaboratoryPress,1989。在过去的几年里,质粒载体已经被用作DNA疫苗用于向细胞体内递送编码抗原的基因。对此它们是特别有利的,因为它们不具有与许多病毒抗体相同的安全考虑。然而,这些与宿主细胞具有兼容启动子的质粒,可以从所述质粒中可操作地编码的基因表达肽。一些通常使用的质粒包括pBR322,pUC18,pUCl9,pRC/CMV,SV40,和pBlueScript。其它的质粒对于本领域的普通技术人员是公知的。另外,可以使用限制性酶和连接反应以移除和增加DNA的特异性片段来定制设计质粒。可以通过各种肠胃外,粘膜和局部途径递送质粒。例如,可以通过肌肉内,皮内,皮下或其它途径注射DNA质粒。其也可以通过鼻内喷雾或滴剂,直肠栓剂和口服施用。其还可以使用基因枪施用到表皮中或粘膜表面。所述质粒可以提供于水溶液,在金颗粒上干燥,或与另一个DNA递送系统联合,所述另一个DNA递送系统包括但不限于脂质体,树状物(dendrimer),螺旋状(cochleate)和微胶囊。
典型地,本发明的CTPS1抑制剂以治疗有效量施用到受试者。
如上文所述的本发明的CTPS1抑制剂的“治疗有效量”表示足够量的化合物。然而,应当理解,本发明的化合物和组合物的总日常用量将由主治医生在合理的医学判断范围内决定。对于任意具体的受试者,具体的治疗有效剂量水平将依赖于各种因素包括治疗的病症以及所述病症的严重性;采用的特定化合物的活性;采用的具体组合物,所述受试者的年龄,体重,总体健康状况,性别和饮食;施用的时间,施用的途径,已经采用的具体化合物的排泄速率;治疗的持续时间;与采用的具体CTPS1抑制剂联合或同时使用的药物;以及在医学领域中公知的类似因素。例如,在本领域的技术内化合物的起始剂量比需要达到所需治疗效果的剂量低,而逐渐增加剂量直到达到所需效果。然而,产品的每日剂量可以在从0.01至1000mg每成年人每天的广泛范围内变化。典型的,所述组合物含有0.01,0.05,0.1,0.5,1.0,2.5,5.0,10.0,15.0,25.0,50.0,100,250和500mg的活性成分,用于待治疗的受试者的剂量症状调整。药物典型地含有从约0.01mg至约500mg的活性成分,典型地从1mg至约100mg活性成分。药物的有效量的剂量水平通常以从0.0002mg/kg至约20mg/kg的体重每天供应,特别是从约0.001mg/kg至7mg/kg体重每天。
本发明的CTPS1抑制剂可以与药学上可接受的赋形剂,以及可选的缓释基质例如可生物降解聚合物联用,以形成药学组合物。
“药学上”或“药学上可接受”指代分子实体和组合物,当其适当的施用到哺乳动物,特别是人时,不产生不利的,过敏的或其它不良反应。药学上可接受的载体或赋形剂指代无毒固体,半固体或液体填充剂,稀释剂,封装材料或任意种类的剂型辅助剂。
在用于口服,舌下,皮下,肌肉内,静脉内,经皮,局部或直肠施用的本发明的药物组合物中,活性成分,单独或与另一种活性成分联合,可以以单位施用形式,作为与常规药物支持物的混合物,施用到动物和人类。适合的单位施用形式包含口服途径形式,例如片剂,凝胶胶囊,粉剂,颗粒剂和口服混悬液或溶液,舌下和颊施用形式,气溶胶,植入物,皮下,经皮,局部,腹膜内,肌肉内,静脉内,真皮下,经皮,鞘内和鼻内施用形式和直肠施用形式。
典型地,所述药物组合物含有媒介物,其对于能够注射的制剂是药学上可接受的。这些媒介物可以具体是等渗的,无菌的,盐溶液(磷酸二氢钠或磷酸氢二钠,钠,钾,钙或镁氯化物和类似物或这些盐的混合物),或干燥的,特别是冷冻干燥的组合物,视情况而定,当添加无菌水或生理盐水时,允许可注射溶液的构建。
适合用于可注射用途的药学形式包括无菌水溶液或分散液;制剂包括芝麻油,花生油,或含水丙二醇;和用于临时制备无菌可注射溶液或分散液的无菌干粉。在所有情况下,所述形式必须是无菌的且必须是流动程度能够容易地注射。其必须在制造和储存的条件下稳定,且必须针对微生物,例如细菌和真菌的污染保存。
包含本发明的化合物作为游离碱(freebase)或药学上可接受的盐的溶液可以在适当地混合了表面活性剂的水中制备,例如,羟丙基纤维素。也可以在丙三醇,液体聚乙二醇,和其混合物和油中制备分散液。在普通的储存和使用条件下,这些制剂含有防腐剂以阻止微生物的生长。
本发明的CTPS1抑制剂可以以中性或盐形式配制到组合物中。药学上可接受的盐包括酸加成盐(使用蛋白的游离氨基形成)和使用无机酸形成的例如,盐酸或磷酸,或那些有机酸例如乙酸,草酸,酒石酸,扁桃酸,和类似物。使用游离羧基形成的盐也可以从无机碱衍生,例如钠,钾,铵,钙,或铁的氢氧化物,和那些有机碱如异丙胺,三甲胺,组氨酸,普鲁卡因(procaine)和类似物。
所述载体也可以是溶剂或分散介质,含有,例如,水,乙醇,多元醇(例如,丙三醇,丙二醇,和液体聚乙二醇,和类似物),其适合的混合物,以及植物油。可以通过,例如使用包被例如卵磷脂,在分散液的情况下维持要求的颗粒大小和通过使用表面活性剂维持适当的流动性。可以通过各种抗生素和抗真菌可溶性试剂带来对于微生物作用的阻止,例如对羟基苯甲酸酯类,氯丁醇,苯酚,山梨酸,硫柳汞,等等。在许多情况下,将优选包括等渗剂,例如,糖或氯化钠。可以通过在所述组合物中使用延迟吸收的试剂带来对所述可注射组合物延长的吸收,例如,单硬脂酸铝和明胶。
通过将所需量的活性多肽整合到适合的溶剂中制备无菌可注射溶液,所述溶剂根据需要具有上文枚举的各种其它成分,接着通过过滤除菌。通常来说,通过将各种无菌活性成分整合到无菌媒介物中制备分散液,所述无菌媒介物含有基本分散介质和所需的上文所枚举的那些其它成分。对于用于制备无菌可注射溶液的无菌粉末,制备的具体方法是真空干燥和冷冻干燥技术,其产出活性成分加上任意另外的所需成分的粉末,来自于其预先无菌过滤的溶液。
一经配置,溶液将以与剂型相符的方式且以治疗有效量施用。所述制剂以各种剂量形式容易地施用,例如上文所述的可注射溶液的类型,但也可以采用药物释放胶囊等等。
对于水溶液中的肠胃外施用,例如,如果必要所述溶液应当适合的缓冲,且所述液体稀释剂首先使用足量的盐水或糖使得等渗。这些具体的水溶液特别适合用于静脉内,肌肉内,皮下和腹膜内施用。在这一点上,可以采用的无菌水介质对于本领域的技术人员根据本公开将是已知的。例如,一个剂量可以溶解于1ml等渗的NaCl溶液并添加到1000ml皮下输注液或注射于输注的建议位点。将依赖于治疗的受试者的情况而必要地发生一些剂量的变化。对施用负责的人将,在任何情况下,确定对于个体受试者的适当剂量。
除了本发明的化合物用于肠胃外施用,例如静脉内或肌肉内注射,其它药学上可接受的形式包括,例如,用于口服施用的片剂或其它固体;脂质体制剂;时间缓释胶囊;和目前使用的任意其它形式。
本发明的CTPS1抑制剂可以与本领域已知的任意免疫抑制剂联合使用。免疫抑制剂包括但不限于,他汀类药物;mTOR抑制剂,例如雷帕霉素或雷帕霉素类似物;TGF-β信号传导剂;TGF-β受体激动剂;组蛋白去乙酰化酶抑制剂,例如曲古抑菌素A;皮质激素类;线粒体功能的抑制剂,例如鱼藤酮;P38抑制剂;NF-κβ抑制剂,如6Bio,地塞米松,TCPA-1,IKKVII;腺苷受体激动剂;前列腺素E2激动剂(PGE2),如米索前列醇(Misoprostol);磷酸二酯酶抑制剂,如磷酸二酯酶4抑制剂(PDE4),如咯利普兰(Rolipram);蛋白酶体抑制剂;激酶抑制剂;G-蛋白偶联受体激动剂;G蛋白偶联受体拮抗剂;糖皮质激素;维甲酸;细胞因子抑制剂;细胞因子受体抑制剂;细胞因子受体激活剂;过氧化物酶体增殖物激活的受体拮抗剂;过氧化物酶体增殖物激活的受体激动剂;组蛋白去乙酰化酶抑制剂;钙调神经磷酸酶抑制剂;磷酸酶抑制剂;PI3KB抑制剂,如TGX-221;自噬抑制剂,例如3-甲基腺嘌呤;芳香烃受体抑制剂;蛋白酶体抑制剂I(PSI);和氧化的ATP,如P2X受体阻滞剂。免疫抑制剂也包括IDO,维生素D3,环孢霉素,例如环孢霉素A,芳基烃受体抑制剂,白藜芦醇,硫唑嘌呤(Aza),6-巯基嘌呤(6-MP),6-硫鸟嘌呤(6-TG),FK506,萨菲菌素A,沙美特罗(salmeterol),霉酚酸酯(MMF),阿司匹林和其它COX抑制剂,尼氟灭酸(niflumicacid),雌三醇(estriol)和雷公藤内酯(triptolide)。在一些实施方案中,本发明的CTPS1抑制剂也可以与抗CD28抗体,IL2拮抗剂或IL15拮抗剂联合使用。
本发明的进一步的方面涉及筛选多种测试物质的方法,所述测试物质有用于在有此需要的受试者中抑制淋巴细胞增殖,所述方法包含由以下项组成的步骤i)测试每种测试物质其抑制CTPS1活性或表达的能力和ii)鉴定抑制CTPS1活性或表达的测试物质从而选择对于在有此需要的受试者中抑制淋巴细胞增殖有用的物质。
本领域已知的任意试验可以用于测试测试物质抑制CTPS1活性的能力。具体地所述试验可以由以下项组成:使用酶的标记的底物和然后确定转化的产物的量。仅要求适当的标记所述底物使得能够通过检测生物合成途径产物中的标记物来检测其转化。所述底物通常是标记的谷氨酰胺或UTP。典型地,所述标记的底物可以是非放射性的或放射性的。例如,在非放射性底物的情况下,可以是C13标记的或氘标记的底物。例如,在放射性底物的情况下,特别是C14标记的或氚标记的底物。典型地,所述标记的底物可以作为水溶液与CTPS1一同添加。所述水溶液中的底物的浓度可以是1μM至1mM。在C14标记的底物的情况下,放射性通常为至少0.1μCi而在3H标记的底物的情况下通常为至少1μCi。使用14-碳或13-碳标记可以是单一的,其中任意一个C位点可能被标记。或者,所述底物可以多重标记的,例如双重,三重,四重或五重。在13-碳标记的情况下,总的碳标记尤为特殊。使用氘或氚的标记可以是单一的或多重的。典型地,所述标记的底物可以酶促或化学制备。所述底物,所述测试物质和所述酶通常孵育足够用于酶促转化的时间。然后可以通过HPLC,薄层色谱法等,将通过所述底物转化产生的CTP从溶液分离。在放射性标记的情况下,可以通过闪烁计数器,通过磷屏成像系统(phosphorimager),通过无线电薄层计数器或通过无线电探测器与层析柱的联合,实现对标记的产物的确定。典型地,HPLC与流动闪烁分析仪(Radiomatic150TR,Packard)的连接使得可以检测色谱峰中的放射性。对于放射性测量,通常将整个样品装载到柱上。通过测量峰高和将它们与标准曲线比较,对标记的产物定量。在非放射性标记的情况下,可以通过NMR光谱(例如,13C-NMR)或质谱(例如,HPLC-MS或GC-MS)常规实现确定。当标记的产物的量比测试物质不存在时确定的标记的产物的量少时,认为测试物质作为CTPS1抑制剂。
在一些实施方案中,在体外试验中探索酶促转化,其中使用表达研究的酶的细胞。在这一具体实施方案中,所述筛选方法包含以下步骤:
a)在培养基中制备表达CTPS1的细胞的悬液,所述培养基用于支持所述细胞的代谢
b)向所述悬液添加预定量的标记的底物,
c)将步骤b)中获得的混合物在预定的温度孵育预定的一段时间
d)从步骤中获得的所述孵育的混合物分离包含标记的产物的部分,通常通过裂解细胞以释放它们的细胞内含物,
e)检测步骤d)中获得的所述部分中标记的产物的浓度,
f)重复步骤b),c),d)和e),在其他方面相同的条件下添加预定量的测试物质,
g)通过观察步骤f)中检测到的标记的产物的浓度是否比步骤e)中检测到的标记的产物的浓度低确定对CTPS1的抑制的存在。
基本上,在步骤e)或f)中检测到的标记的产物的浓度代表了CTP库。当细胞与测试物质孵育时CTP库的减少提示所述测试物质是CTPS1抑制剂。
在体外试验中可以使用各种细胞。典型地所述细胞是天然表达CTPS1的T细胞。在一些实施方案中,可以利用各种各样的宿主表达载体系统来在感兴趣的细胞中表达CTPS1。这些系统包括但不限于,哺乳动物细胞系统例如人细胞系。所述哺乳动物细胞系统可以包容重组表达构建物,其含有来源于哺乳动物细胞基因组或哺乳动物病毒的启动子(例如,腺病毒晚期启动子或疫苗病毒7.5K启动子)。可以通过几种本领域已知的方法将待分析的DNA编码蛋白(例如,CTPS1)瞬时或稳定地表达在所述细胞系中,所述方法例如磷酸钙介导的,DEAE-葡聚糖介导的,脂质体介导的,病毒介导的,电穿孔介导的和显微注射递送。根据DNA,细胞系,以及后续采用的试验类型,这些方法的每一种可能要求对组合的实验参数优化。此外,可以使用天然携带并表达目标蛋白的核酸序列的原生细胞系。
在本领域中公知的试验也可以用于确定测试物质是否能够抑制CTPS1的表达。典型的,将表达CTPS1的细胞群体在测试物质存在下培养,接着确定CTPS1的表达水平并与所述测试物质不存在时确定的水平比较。当在所述测试物质存在时确定的CTPS1表达水平比所述测试物质不存在时确定的CTPS1表达水平低,可以得出结论所述测试物质是CTPS1抑制剂。
可以通过各种技术进行对基因表达水平的确定。一般来说,确定的表达水平是相对表达水平。更典型地,所述确定包含将样品与选择的试剂接触,例如探针,引物或配体,从而检测样品中本来的感兴趣的多肽或核酸的存在,或测量它们的量。可以在任意适合的装置中进行接触,例如板,微量滴定盘,试管,孔,玻璃,柱,等等。在一些实施方案中,所述接触在使用试剂包被的基质上进行,例如核酸阵列或特异性配体阵列。所述基质可以是固体或半固体基质,例如包含玻璃,塑料,尼龙,纸,金属,聚合物等等的任意适合的支持物。所述基质可以是各种形式和大小,例如载玻片,膜,珠,柱,凝胶,等等。所述接触可以在适合形成可检测复合物的任意条件下进行,例如核酸杂交或抗体-抗原复合物,所述可检测复合物在所述试剂和所述样品的核酸或多肽之间形成。
在一些实施方案中,可以通过确定mRNA的量确定表达水平。
用于确定mRNA的量的方法是本领域公知的。例如,首先根据标准方法提取样品(例如,从受试者制备的细胞或组织)中含有的核酸,例如使用分解酶或化学溶液,或通过核酸结合树脂遵循制造商的说明提取。接着通过杂交(例如,Northern印记分析)和/或扩增(例如,RT-PCR)检测提取的mRNA。典型地具体是定量或半定量RT-PCR。实时定量或半定量RT-PCR是特别有利的。
其它的扩增方法包括连接酶链式反应(LCR),转录介导的扩增(TMA),链置换扩增(SDA)和基于核酸序列的扩增(NASBA)。
具有至少10个核苷酸并展现出与本文感兴趣的mRNA序列互补性或同源性的核酸可用作杂交探针或扩增引物。应当理解这些核酸不需要相同,但通常与可比大小的同源性区域至少约80%相同,更典型地85%相同和甚至更典型地90-95%相同。在某些实施方案中,使用核酸与适合的工具,例如可检测标签联合用于检测杂交将是有利的。许多各种不同的适合的指示物是本领域已知的,包括荧光的,放射性的,酶促的或其它配体(例如,亲和素/生物素)。
探针通常包含长度为10至1000个核苷酸的单链核酸,例如10和800个,更典型地15和700个,典型地20和500个。引物通常是较短的单链核酸,长度为10至25个核苷酸,设计为完美地或近乎完美地与感兴趣的,待扩增的核酸匹配。所述探针和引物对于它们杂交的核酸是“特异性的”,即它们通常在高严格杂交条件下杂交(对应最高的解链温度Tm,例如,50%甲酰胺,5x或6xSCC。SCC为0.15MNaCl,0.015M柠檬酸钠)。
在上述扩增和检测方法中使用的核酸引物或探针可以组装为试剂盒。这种试剂盒包括通用引物和分子探针。具体的试剂盒还包括必要的组分以确定扩增是否发生。所述试剂盒还可以包括,例如,PCR缓冲液和酶;阳性对照序列,反应对照引物;和用于扩增和检测特异性序列的说明书。
在一些实施方案中,通过DNA芯片分析确定表达水平。这种DNA芯片或核酸微阵列由不同的核酸探针组成,其与基质化学附接,所述基质可以是微芯片,玻璃载玻片或微球大小的珠子。微芯片可以由聚合物,塑料,树脂,多糖,二氧化硅,基于二氧化硅的材料,碳,金属,无机玻璃,或硝化纤维构成。探针包含核酸例如cDNA或寡核苷酸,其可以是约10至约60个碱基对。为了确定表达水平,来自于测试受试者的样品,可选的首先进行逆转录,被标记并在杂交条件下与微阵列接触,引起目标核酸和附接到所述微阵列表面的探针序列之间形成复合物。接着检测标记的杂交复合物并可以定量或半定量。可以通过多种方法完成标记,例如通过使用放射性或荧光标记。本领域的技术人员可以使用多种微阵列杂交技术的变体(见,例如Hoheisel的综述,NatureReviews,Genetics,2006,7:200-210)。
用于确定所述基因的表达水平的其它方法包括确定由所述基因编码的蛋白的量。这种方法包含将生物样品与能够选择性的与样品中存在的标志物蛋白相互作用的结合伴侣接触。所述结合伴侣通常是抗体,其可以是多克隆或单克隆的,典型地是单克隆的。
可以使用标准的电泳和免疫诊断技术检测蛋白的存在,包括免疫试验例如竞争,直接反应,或夹心类型的试验。这些试验包括但不限于,Western印记;凝集测试;酶标记和介导的免疫试验,例如ELISA;生物素/亲和素类型的试验;放射免疫试验;免疫电泳;免疫沉淀,等等。所述反应通常包括揭示标签例如荧光,化学发光,放射性,酶标签或染料分子,或用于检测抗原和与其反应的一种或多种抗体之间的复合物形成的其它方法。
前述试验通常包含将液相中未结合的蛋白从抗原-抗体复合物结合的固相支持物分离。可以用于本发明的实施的固相支持物包括基质例如硝化纤维(例如,以膜或微量滴定孔的形式);聚氯乙烯(例如,薄片或微量滴定孔);聚苯乙烯胶乳(例如,珠子或微量滴定板);聚偏氟乙烯(polyvinylidinefluoride);重氮化纸;尼龙膜;活化珠,磁响应珠等等。
更具体的,可以使用ELISA方法,其中使用针对待测试蛋白的抗体包被微量滴定板的孔。接着将含有或怀疑含有标志物蛋白的生物样品添加到所述包被的孔。在孵育一段足够允许形成抗体-抗原复合物的时间后,可以洗涤所述板以去除未结合的部分并添加可检测地标记的第二结合分子。所述第二结合分子允许与任意捕获的样品标志物蛋白反应,洗涤板,使用本领域公知的方法检测所述第二结合分子的存在。
典型地,所述测试物质可以选自下组:肽,模拟肽,小有机分子,抗体,适体或核酸。例如,根据本发明的测试物质可以选自预先合成的化合物库,或其结构已经在数据库中确定的化合物库,或来自于已经从头合成的化合物库。在一些实施方案中,所述测试物质可以选自小有机分子。如本文所使用的,术语“小有机分子”指代大小与通常用于药物的那些有机分子可比的分子。该术语排除生物大分子(例如,蛋白,核酸等);具体的小有机分子的大小范围高达2000Da,而最典型地高达约1000Da。
本发明的筛选方法非常简单。它可以使用大量的测试物质连续地或并列地进行。所述方法可以容易地适配机器人。例如,可以使用高通量筛选技术进行以上试验,用于鉴定测试物质用于开发可能对于炎症性肠病的治疗或预防有用的药物。为了使用自动化机器人系统实现多重试验,可以使用多孔板(例如,96-,389-,或1536孔板)进行高通量筛选技术。因此,可以以高效的方式分析测试物质的大型库。用于鉴定测试物质的具体策略开始于使用融合了任意基因的启动子的报告基因转染的培养细胞,所述基因通过应激反应通路激活。更具体的,在微量滴定板(96孔或384孔)中生长的稳定转染的细胞可适于化合物库的高通量筛选。所述库中的化合物以自动化的方式每次一个应用到所述微量滴定盘的孔,其含有如上文所述的转基因细胞。一旦鉴定了激活一个目标基因的测试物质,接着优选确定它们在整合的应激反应通路中的作用位点。确定作用位点对于开发更精确的试验用以优化所述目标物质是特别有用的。
在一些实施方案中,已经阳性选择的测试物质可以进行进一步的选择步骤,基于进一步在体外试验或动物模型生物体中分析其特性,例如啮齿动物模型系统,用于在人类中使用前所需的治疗活性。
例如,体外试验可以包括使用B细胞系或T细胞系例如Jurkat细胞系,或MOLT-4细胞系。具体的,所述方法可以进一步包含由以下项组成的步骤:提供B或T细胞系,使得所述细胞系与选择的测试物质接触,确定所述B或T细胞系的增殖水平,将所述增殖水平与所述测试物质不存在时确定的增殖水平比较,和当所述测试物质存在时确定的增殖水平比所述测试物质不存在时确定的增殖水平低时,阳性选择所述测试物质。
例如,可以用于确定施用所选CTPS1抑制剂是否指示的试验,包括细胞培养试验其中受试者组织样品在培养物中生长,并暴露于或否则与CTPS1抑制剂接触,以及观察这种组合物对所述组织样品的影响。所述组织样品可以通过活组织检查从所述受试者获得。这一测试允许鉴定治疗上最有效的CTPS1抑制剂。在各种具体的实施方案中,可以使用参与自身免疫的细胞类型(例如,T细胞)的代表细胞实施体外试验,以确定测试物质对于这些细胞类型是否具有所需的效果。
可以使用任意公知的动物模型用于探索筛选的CTPS1抑制剂的体内治疗效果。例如,可以通过使用本领域已知的各种炎症性关节炎的实验动物模型确定筛选的CTPS1抑制剂的治疗活性,所述模型描述于CroffordL.J.和WilderR.L.,"ArthritisandAutoimmunityinAnimals",inArthritisandAlliedConditions:ATextbookofRheumatology,McCartyetal.(eds.),Chapter30(Lee和Febiger,1993)。也可以使用炎症性关节炎和自身免疫风湿病的实验和自发动物模型来评估筛选的CTPS1抑制剂的抗炎症活性。可以使用本领域技术人员已知的标准技术监控/评估CTPS1抑制剂减少一种或多种自身免疫疾病的症状的效果。可以通过,例如从所述哺乳动物获得外周血样品,使用例如Ficoll-Hypaque(Pharmacia)梯度离心将淋巴细胞从外周血的其它组分例如血浆分离,以及使用台盼蓝对淋巴细胞计数来确定哺乳动物中的外周血淋巴细胞计数。可以通过,例如使用例如Ficoll-Hypaque(Pharmacia)梯度离心将淋巴细胞从外周血的其它组分例如血浆分离,使用缀合到FITC或藻红蛋白的针对T细胞抗原例如CD2,CD3,CD4,和CD8的抗体标记T细胞,以及通过FACS测量T细胞的数量来确定哺乳动物中外周血T细胞的计数。此外,可以使用本领域技术人员已知的技术例如FACS,确定对具体的T细胞亚群的效果(例如,CD2+,CD4+,CD8+,CD4+RO+,CD8+RO+,CD4+RA+,或CD8+RA+细胞)。因此可以容易地评估所述动物模型中的淋巴细胞增殖。可用于体内筛选的动物模型的其它例子包括用于脑脊髓炎EAE的动物,或lpr小鼠。
将通过以下附图和实施例进一步说明本发明。然而,这些实施例和附图不应当以任何方式解释为限制本发明的范围。
附图:
图1.响应TCR-CD3活化的T细胞增殖需要CTPS1。a,T细胞增殖,其中使用含有针对CTPS1的shRNA(ShCTPS1#1或ShCTPS1#2)或含有混杂shRNA(Sh混杂)的载体沉默CTPS1表达,所述载体具有GFP报告基因。GFP+细胞的代表性点图对应转导的细胞(左上图)。紫色染料稀释的代表性柱状图显示刺激后的细胞分裂(左下图)。曲线显示长期扩增中重复刺激后GFP+转导细胞的百分比(中间图)。转导的细胞中对CTPS1和CRPS2表达的免疫印迹(右图)。ACTIC作为内参。一个代表两次实验。b,c,通过空载体或含有野生型CTPS1的载体转导的对照(Ctr.)和CTPS1缺陷的T细胞(患者P1.2)的增殖。紫色染料稀释(b,左图)和刺激后的细胞分裂指数(b,右图)的代表性柱状图。一式三份的均值和s.d.,一个代表两次实验。曲线示出和(a)中相同的GFP+转导的细胞百分比(c,左图)。代表性数据来自2次独立实验的一个。和(a)中相同的免疫印迹(c,右图)。d,紫色染料稀释的代表性柱状图,示出对照(Ctr.)和CTPS-1缺陷的细胞(患者P1.2)的细胞分裂。刺激前,使用所示的核苷或核苷酸孵育细胞。数据来自3次独立实验的一个。e,与(d)相同除了在刺激前和刺激期间使用去氮尿苷孵育对照T细胞。数据来自3次独立实验的一个代表性实验。f,来自健康对照(Ctr.)的原始T细胞和CTPS1缺陷细胞(患者1.2)在使用抗CD3/CD28包被的珠子刺激后的细胞提取物中的CTP浓度。对照细胞在刺激之前和期间使用或不使用去氮尿苷孵育。数据来自3次独立的实验。g,来自健康对照(Ctr.)和CTPS1缺陷的患者(Pat.)的EBVB细胞系的细胞提取物中的CTP的浓度,所述细胞使用或不使用含有野生型CTPS1的载体转导。P1.1(正方形),P1.2(圆形)和P2.1(三角形)。对于对照,符号对应不同的供体细胞。数据来自于2次独立的实验。未配对t检验。***P<0.001。h,通过空载体或含有野生型CTPS的载体转导的CTPS-1缺陷的EBVB细胞系(P1.2和P2.1)的增殖。曲线示出了培养物中GFP+转导的细胞百分比。
实施例:人类中CTP合酶1缺陷揭示了其在淋巴细胞增殖中的中心作用
方法:
从供体,患者和患者家属获得了知情同意。该研究和方案符合1975年Helsinki宣言以及当地法律和道德准则。从外周血细胞提取的基因组DNA用于全外显子组测序(Illumina)和CTPS1测序。Ficoll纯化PBMC并使用植物血凝素(PHA)活化3天,接着在RPMI培养基中培养,所述培养基使用5%的AB型人血清和IL-2(100UI/ml)补充。使用各种有丝分裂原再刺激原始T细胞并通过免疫印迹分析记忆和活化标志物,钙通量,细胞因子分泌,凋亡和TCR-CD3信号传导级联分子。使用针对CTPS1蛋白中间残基(341至355)产生的抗体(#SAB111071,Sigma),通过免疫印记检测CTPS1蛋白。对于增殖和细胞周期和试验,细胞被剥夺IL-2持续3天,在使用抗CD3抗体(1g.ml-1)单独(克隆OKT3,eBiosciences)或与CD3/CD28包被的珠子(Invitrogen)一起再刺激之前,使用CellTrace紫色染料(Invitrogen)或5-乙炔基-2’-脱氧尿苷(EdU)(Click-IT,Invitrogen)孵育。96小时后,通过监控CellTrace紫色染料标记稀释评估增殖。通过测量40小时期间EdU整合到新合成的DNA中确定细胞周期。使用Flowjo软件(BDBiosciences)计算分裂指数。对于通过shRNA表达(Openbiosystems)的CTPS1基因沉默,在PHA刺激的第三天转导细胞。对于补救实验(rescueexperiments),使用含有CTPS1基因的慢病毒载体(Invitrogen)转导CTPS1缺陷的细胞,并在转导后5天时进行增殖。在长期扩增中,使用CD3/CD28包被的珠子每48小时重复的刺激细胞,且每24小时确定培养物中转导的GFP+细胞。通过液相色谱-质谱(UPLC-xevoTQS,Waters)测量细胞内的核苷酸含量。使用PRISM软件(GraphPad)通过双尾Student’st检验计算P值。
结果
我们最初研究了来自英格兰西北地区的两个不相关的家庭(家庭1和2),他们的四个孩子患有严重的和复发的Epstein-Barr病毒(EBV)感染,在他们中已经排除了已知的原发性免疫缺陷10(表1)。此后,从测试的具有严重EBV感染的34位患者(33个家庭)鉴定了来自三个不相关的家庭(家庭3至5)的另外四位患者。所有的患者具有严重慢性病毒感染的早期发作,主要由疱疹病毒引起,包括EBV和水痘带状疱疹病毒(VZV)并且也患有复发的封装细菌感染,其为典型特征是适应性免疫组合缺陷的一系列感染(CID)11(表1)。总的来说,临床表型严重,因为一位患者在4岁时死于散播性VZV感染而6位患者在生命的第一年经历了造血干细胞移植(HSCT)。值得注意的是,没有一位患者具有外造血表现(extra-hematopoieticmanifestations)(表1)。
免疫学研究表明患者具有倒转的CD4:CD8T细胞比率,正常或升高的免疫球蛋白水平,其中在大多数患者中具有增加的IgG但低IgG2水平,对肺炎链球菌(Streptococcuspneumoniae)具有低抗体滴度和可变的淋巴细胞减少(lymphopenia),其在感染发作期间恶化,而其它血细胞计数通常正常。在患者P1.2中进行了进一步的分析,其表现出天然的CD4+T细胞淋巴细胞减少,增加的效应记忆T细胞数量,低数量的记忆CD27+B细胞,完全不存在两种不变的T细胞群体(CD3+V24+V11+)iNKT和(CD3+CD161高V7.2+)MAIT细胞,以及受损的PHA-和抗原诱导的外周血单核细胞(PBMCs)增殖。
为了鉴定这些患者中免疫缺陷根本的基因缺陷,我们在三位患者(P1.1,P1.2和P2.1)中进行了全外显子组测序(WES)。在三位患者中发现的遗传变异的交集指向编码CTP合酶1的CTPS1基因中独特和共同的纯合G到C突变,在染色体1中的位置41475832,其在dbSNP数据库中具有分配的rsID(rs145092287)。CTPS1包含19个外显子,其编码含有CTP合成酶结构域和谷氨酰胺酰胺转移结构域的67-kDa蛋白,促进从UTP和谷氨酰胺形成CTP12。鉴定的所述突变影响内含子17-18和外显子18的连接处的剪接供体位点(IVS18-1G>C),导致表达缺少外显子18的异常转录物。发现这一剪接突变体是有害的,因为在来自患者的EBV-转化的B细胞和原始T细胞的裂解物中,通过使用两种不同的抗CTPS1抗体不能检测到CTPS1蛋白的表达。相反的,在患者的细胞裂解物中,CTPS2正常表达。来自相同地理区域具有相似临床表现的另外四位患者也发现对于相同的剪接突变是纯合的(表1)。在受到影响的五个家庭中,所有的亲本对于所述突变是杂合的,且测试的健康兄弟姐妹对于IVS18-1G>C突变也是杂合的。对来自英格兰西北部的752名健康个体的测序鉴定了两名对于IVS18-1G>C突变杂合的个体,对应估算的纯合子频率为1:560,000。这代表了与从可得的外显子组数据库估计的频率相比大于10倍的增加。通过WES在P1.1,P1.2和P2.1中发现的纯合区域以及在所有患者中分析多态性微卫星标记物揭示它们共有IVS18-1G>C突变周围1.1Mb相同的纯合区域。所有这些数据都指示了奠基者(founder)效应。这些观察使得我们得出以下结论:这些患者中由于CTPS1突变导致的免疫缺陷可能主要与T细胞免疫缺陷相关。
我们接着在正常组织中检测了CTPS1表达。不同组织间的CTPS1mRNA表达是可比的,除了在T细胞中CTPS1的表达在响应TCR-CD3和CD28共刺激的细胞活化后强烈上调。有趣的是,在原始T细胞和来自PBMC的T细胞的裂解物中,CTPS1蛋白几乎不可检测。相反的,CTPS2的表达可以容易地检测。通过抗CD3抗体或佛波醇12-肉豆蔻酸酯13-乙酸(PMA)和离子霉素刺激诱导的T细胞活化诱导CTPS1蛋白表达,而使用IL-2和/或IL-15激活仅导致较弱的效果。在相同的实验条件下,也诱导CTPS2的表达但程度较小。在TCR-CD3刺激的原始T细胞中,作为CTPS1基因转录激活的结果,CTPS1蛋白的表达从12小时增强并持续至96小时。正如所预料的,在来自CTPS1缺陷的患者(P1.2)的原始T细胞中没有检测到CTPS1的表达,这与CTPS1mRNA的检测形成对比并提示蛋白的不稳定。这些数据表明,通过TCR的T细胞活化导致快速并持续的CTPS1蛋白表达。值得注意的是,在通过抗BCR和CpG,IL4和CD40L或PMA和离子霉素激活的B细胞中,也发现CTPS1上调。
为了进一步表征T细胞中CTPS1缺陷的结果,我们研究了近端T细胞活化信号以及后期响应。在使用抗CD3抗体刺激来自患者P1.2的T细胞后,CTPS1缺陷的细胞展现出正常的总体蛋白酪氨酸磷酸化模式和正常的PKC-θ,PLCγ-1,IκBα和NFAT2c磷酸化,除了发现ERK1/2磷酸化降低。此外,发现Ca++通量和后期响应例如去颗粒作用(degranulation)和细胞因子产生正常,虽然CD25和CD69的上调显著减少。我们还注意到CTPS1缺陷的原始细胞与对照细胞相比,展现出少量但显著的基底和活化诱导的细胞死亡的增加。总的来说,这些数据提示CTPS1缺陷对于TCR-CD3下游的信号传导影响有限。
由于CTP库是DNA合成的潜在限制因子8,13,我们仔细的分析了CTPS1缺陷的T细胞的增殖。响应通过抗原、抗CD3抗体、或抗CD3和抗CD28抗体共刺激的活化,来自三位患者(P1.1,P1.2和P2.2)的CTPS1缺陷的细胞未能维持增殖反应,如通过H3-胸苷摄取和CFSE或紫色细胞示踪染料稀释所测量的(产生细胞增殖的弱指数)。在活化的CTPS1缺陷的T细胞中,也发现对3H-尿苷和3H-胞苷的摄取减弱,提示RNA合成受到影响。当伴随测试时,通过3H-亮氨酸摄取确定了蛋白合成也减少。CTPS1缺陷细胞的缺陷增殖也伴随着细胞周期进展的缺乏,因为大多数细胞被阻滞在G1期。因为其活化后B淋巴细胞中CTPS1的表达也增加,通过抗BCR和CpG活化也检查了CTPS1缺陷的B细胞的增殖,揭示其增殖的阻塞,而IL-2激活的NK细胞的增殖似乎受到的影响较小。
通过慢病毒与GFP报告基因一起转导两种不同的shRNA,在对照T细胞中下调CTPS1的表达,导致了GFP阳性细胞的CD3介导的增殖特异性减少。在非目标GFP阴性的细胞,或在使用混杂shRNA的细胞靶标中,没有检测到增殖变化。由于对CTPS1表达的抑制导致的增殖减少引起选择性的细胞生长不利,随着时间GFP靶向的细胞数量减少(中间图)。在其中CTPS1表达下调的JurkatT细胞中,也观察到了相似的增殖率下降。
总的来说,这些结果表明CTPS1缺陷导致响应TCR-CD3活化的T细胞增殖的缺陷。为了正式地证实CTPS1缺陷和缺陷的T细胞增殖之间的因果关系,我们使用野生型CTPS1或直接添加CTP或其胞苷前体(其通过补救途径作用于CTP水平)实施了重构实验。在CTPS1缺陷的T细胞中,异位CTPS1的表达完全恢复了当CD3刺激时的增殖,并使得细胞能够选择性的扩增,如通过表达CTPS1的GFP阳性细胞的积累所示。在使用空载体转导的CTPS1缺陷细胞或使用含有CTPS1的载体转导的对照细胞中,没有检测到这种效果。
通过添加CTP或胞苷,CTPS1缺陷细胞的增殖和CD25表达也恢复到了正常水平。相反,添加UTP,GTP和ATP或尿嘧啶,鸟嘌呤和腺嘌呤的混合物不引起CTPS1缺陷细胞的增殖增加。去氮尿苷,一种UTP类似物和已知的CTP合成酶活性抑制剂14,完全阻断了对照细胞响应CD3活化的T细胞增殖,而不影响近端TCR-CD3介导的响应,与CTPS1缺陷细胞中观察到的结果类似。正如所预期的,去氮尿苷对T细胞增殖的抑制通过添加CTP完全回复,且通过添加UTP部分回复,但不通过添加ATP或GTP回复。对活化的CTPS1缺陷原始T细胞和CTPS1缺陷B/EBV细胞系中核苷酸库的分析揭示了CTP水平的降低,正如在使用去氮尿苷处理的活化正常细胞中所观察到的。缺陷的CTPS1表达或添加去氮尿苷也引起了活化的T细胞中ATP,GTP和UTP库的减少,提示了核苷酸库中的相互联系15。相反,在静息的CTPS1缺陷T细胞中,发现CTP水平以及ATP,GTP和UTP水平正常或增加,因为在静息细胞中补救途径占主要地位16。在CTPS1缺陷的B/EBV细胞系中,野生型CTPS1的表达将CTP恢复到与对照细胞可比的水平,并赋予细胞在培养物中选择性细胞生长的优势。
本研究揭示了CTPS1在促进人T细胞在它们活化后增殖中的关键作用。然而,发现B细胞的增殖也依赖于CTPS1。这可能直接参与在CTPS1缺陷患者中看到的对封装细菌感染的易感性,且导致了肺炎链球菌抗体的低滴度,其是独立于T细胞的B细胞反应。CTPS1在B细胞中的作用可能与在T细胞中发现的不同或/和较不重要。值得注意的是,CTPS1缺陷的B细胞当通过EBV转化时保存了完整的扩增能力,并且患者具有正常的Ig水平和/或升高的IgG。NK细胞的扩增减少和iNKT和MAIT细胞的低数量也可能有助于CTPS1免疫缺陷,因为已经提出这些细胞在范围广泛的免疫反应中发挥作用,包括抗EBV反应17-20。CTPS1缺陷不导致其他的显著临床结果这一发现支持其它细胞谱系和组织中CTPS2活性的冗余。有趣的是,之前已经证明了在PHA刺激的细胞中包括CTP的嘧啶池通过从头合成途径强烈扩增,包括增加的CTPS活性8,9。本文报道的在活化的T细胞中对CTPS1表达的诱导似乎是CTP库增加的主要决定因素。与这些数据一致,通过向CTPS1缺陷的T细胞添加CTP将增殖恢复到了正常水平。TCR信号传导通过什么机制诱导T细胞中CTPS1的快速表达仍然待确定。有趣的是注意到CTPS1缺陷似乎不严重减弱T细胞分化,提示胸腺细胞中的CTP库可能来自于核苷补救途径和/或CTPS2活性8,21-23。然而,值得注意的是CTPS1活性对于由抗原刺激诱导的强烈的细胞分裂是关键性的,如由病毒感染期间CD8+T细胞的大量增殖和扩增所示例的24,25。
最近,显示嘧啶的从头合成途径依赖于活化嘧啶合成的第一酶促步骤的mTORC1和S6蛋白(S6K)激酶的转录后调节26-28。因此,不同的调节机制控制嘧啶的从头合成。基于本研究,淋巴细胞中CTPS1介导的对CTP合成的调整似乎是促成适应性免疫反应的关键要素。考虑到CTPS1缺陷表型的淋巴细胞特异性,CTPS1特异性抑制剂将是潜在的高特异性免疫抑制药物,能够抑制自身或同种异体特异性T和B细胞反应,而没有另外的毒性。总之,我们的结果提供了嘧啶从头合成途径作为当通过抗原激活时T和B淋巴细胞增殖的关键步骤的作用的第一个体内证据。
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Claims (27)
1.一种用于在有此需要的受试者中减少或抑制淋巴细胞增殖的方法,包括向所述受试者施用治疗有效量的至少一种CTP合酶1(CTPS1)抑制剂。
2.权利要求1的方法,用于抑制或减少T细胞增殖。
3.权利要求1的方法,用于抑制或减少B细胞增殖。
4.权利要求1的方法,其中所述受试者是经移植的受试者。
5.权利要求1的方法,其中所述受试者使用选自下组的移植物移植:心脏,肾脏,肺,肝,胰腺,胰岛,脑组织,胃,大肠,小肠,角膜,皮肤,气管,骨,骨髓,肌肉,或膀胱。
6.权利要求1的方法,用于防止或抑制接受体受试者对供体组织,细胞,移植物,或器官移植的排斥相关的免疫反应。
7.权利要求1的方法,用于防止接受体中对移植物的急性排斥和/或用于长期维持治疗来防止接受体中对移植物的排斥。
8.权利要求1的方法,用于防止宿主抗移植物病(HVGD)或移植物抗宿主病(GVHD)。
9.根据权利要求1使用的CTPS1抑制剂,其中所述受试者患有自身免疫疾病或淋巴增生性疾病或是经移植的受试者。
10.权利要求4的方法,其中所述CTPS1抑制剂在移植之前和/或之后定期施用到所述受试者。
11.权利要求1的方法,其中所述受试者患有自身免疫疾病。
12.权利要求11的方法,其中所述自身免疫疾病选自下组:阿狄森氏病(Addison'sDisease),过敏,斑秃(AlopeciaAreata),阿尔兹海默病,抗中性粒细胞胞浆抗体(ANCA)相关性血管炎,强直性脊柱炎,抗磷脂综合征(休斯综合症(HughesSyndrome)),关节炎,哮喘,动脉粥样硬化,动脉粥样硬化斑块,自身免疫性疾病(例如,狼疮,RA,MS,格雷夫斯病(Graves'disease)等),自身免疫性溶血性贫血,自身免疫性肝炎,自身免疫性内耳病,自身免疫性淋巴增生综合征,自身免疫性心肌炎,自身免疫性卵巢炎,自身免疫性睾丸炎,无精子症,贝切特氏病(Behcet'sDisease),贝格尔氏病(Berger'sDisease),大疱性类天疱疮,心肌病,心血管疾病,乳糜泻/腹腔疾病(CeliacSprue/Coeliacdisease),慢性疲劳免疫功能障碍综合征(CFIDS),慢性特发性多发性神经炎,慢性炎性脱髓鞘性,多发性神经根性神经病(Polyradicalneuropathy)(CIPD),慢性复发性多发性神经病(格林-巴利综合征(Guillain-Barrésyndrome)),丘-斯综合征(Churg-StraussSyndrome,CSS),瘢痕性类天疱疮,冷凝集素疾病(CAD),慢性阻塞性肺病(COPD),CREST综合征,克罗恩氏病(Crohn'sdisease),皮炎,疱疹样皮炎(Herpetiformus),皮肌炎,糖尿病,盘状狼疮(DiscoidLupus),湿疹,获得性大疱性表皮松解症(Epidermolysisbullosaacquisita),原发性混合型冷球蛋白血症(EssentialMixedCryoglobulinemia),埃文氏综合征(Evan'sSyndrome),眼球突出,纤维肌痛,肺出血肾炎综合征(Goodpasture'sSyndrome),桥本氏甲状腺炎(Hashimoto'sThyroiditis),特发性肺纤维化,特发性血小板减少性紫癜(ITP),IgA肾病,免疫增生性疾病或病症(例如,银屑病),炎性肠病(IBD),包括克罗恩病和溃疡性结肠炎,胰岛素依赖型糖尿病(IDDM),间质性肺病,幼年型糖尿病,幼年型关节炎,幼年特发性关节炎(JIA),川崎氏病(Kawasaki'sDisease),兰伯特-伊顿肌无力综合征(Lambert-EatonMyasthenicSyndrome),扁平苔藓,狼疮,狼疮性肾炎,淋巴细胞性垂体炎(LymphoscyticLypophisitis),梅尼埃病(Ménière'sDisease),米勒费希尔综合症(MillerFisherSyndrome)/急性播散脑脊髓脊神经根病,混合型结缔组织病,多发性硬化症(MS),肌肉风湿,肌痛性脑脊髓炎(ME),重症肌无力,眼部炎症,落叶型天疱疮(PemphigusFoliaceus),寻常型天疱疮(PemphigusVulgaris),恶性贫血,结节性多动脉炎,多软骨炎,多腺体综合征(惠特克氏综合征(Whitaker'ssyndrome)),风湿性多肌痛,多发性肌炎,原发性无丙种球蛋白血症,原发性胆汁性肝硬化/自身免疫性胆管病(Autoimmunecholangiopathy),银屑病,银屑病关节炎,雷诺氏现象(Raynaud'sPhenomenon),莱特尔氏综合征(Reiter'sSyndrome)/反应性关节炎,再狭窄,风湿热,风湿性疾病,类风湿关节炎,结节病,施密特综合征(Schmidt'ssyndrome),硬皮病,综合症,僵人综合症(Stiff-ManSyndrome),系统性红斑狼疮(SLE),系统性硬皮病,大动脉炎,颞动脉炎/巨细胞动脉炎,甲状腺炎,1型糖尿病,2型糖尿病,溃疡性结肠炎,葡萄膜炎,血管炎,白癜风和韦格纳氏肉芽肿(Wegener'sGranulomatosis)。
13.权利要求1的方法,其中所述CTPS1抑制剂是氨基酸谷氨酰胺的任意功能性类似物,衍生物,取代产物,同分异构物,或同源物,其保留了谷氨酰胺结合CTPS1抑制剂的特性。
14.权利要求1的方法,其中所述CTPS1抑制剂是正亮氨酸衍生物,例如6-重氮-5-氧-L-正亮氨酸(DON)。
15.权利要求1的方法,其中所述CTPS1抑制剂是阿西维辛(acivicin)。
16.权利要求1的方法,其中所述CTPS1抑制剂是UTP的类似物。
17.权利要求16的方法,其中所述CTPS1抑制剂是去氮尿苷。
18.权利要求1的方法,其中所述CTPS1抑制剂选自下组:环戊烯基胞嘧啶(CPEC),吉西他滨(Gemcitabine)(2',2'-二氟脱氧胞苷,dFdC),放线菌素D,环己酰亚胺,联丁酰基环AMP,和6-氮杂尿苷。
19.权利要求1的方法,其中所述CTPS1抑制剂是CTPS1表达的抑制剂。
20.权利要求19的方法,其中所述CTPS1抑制剂是siRNA或反义寡核苷酸。
21.权利要求1的方法,其中所述CTPS1抑制剂与至少一种免疫抑制剂联用。
22.权利要求21的方法,其中所述免疫抑制剂选自下组:他汀类药物;mTOR抑制剂,例如雷帕霉素或雷帕霉素类似物;TGF-β信号传导药剂;TGF-β受体激动剂;组蛋白去乙酰化酶抑制剂,例如曲古抑菌素A;皮质激素类;线粒体功能的抑制剂,例如鱼藤酮;P38抑制剂;NF-κβ抑制剂,如6Bio,地塞米松,TCPA-1,IKKVII;腺苷受体激动剂;前列腺素E2激动剂(PGE2),如米索前列醇(Misoprostol);磷酸二酯酶抑制剂,如磷酸二酯酶4抑制剂(PDE4),如咯利普兰(Rolipram);蛋白酶体抑制剂;激酶抑制剂;G-蛋白偶联受体激动剂;G蛋白偶联受体拮抗剂;糖皮质激素;维甲酸;细胞因子抑制剂;细胞因子受体抑制剂;细胞因子受体激活剂;过氧化物酶体增殖物激活受体拮抗剂;过氧化物酶体增殖物激活的受体激动剂;组蛋白去乙酰化酶抑制剂;钙调神经磷酸酶抑制剂;磷酸酶抑制剂;PI3KB抑制剂,如TGX-221;自噬抑制剂,例如3-甲基腺嘌呤;芳香烃受体抑制剂;蛋白酶体抑制剂I(PSI);和氧化的ATP,如P2X受体阻断剂。免疫抑制剂也包括IDO,维生素D3,环孢霉素,例如环孢霉素A,芳基烃受体抑制剂,白藜芦醇,硫唑嘌呤(azathiopurine)(Aza),6-巯基嘌呤(6-MP),6-硫鸟嘌呤(6-TG),FK506,萨菲菌素(sanglifehrin)A,沙美特罗(salmeterol),霉酚酸酯(MMF),阿司匹林和其它COX抑制剂,尼氟灭酸(niflumicacid),雌三醇(estriol)和雷公藤内酯(triptolide)。
23.权利要求1的方法,其中本发明的CTPS1抑制剂与抗CD28抗体,IL2拮抗剂或IL15拮抗剂联用。
24.一种筛选可用于在有此需要的受试者中抑制淋巴细胞增殖的多种测试物质的方法,包含由以下项组成的步骤:i)测试每种测试物质抑制CTPS1活性或表达的能力,和ii)鉴定抑制CTPS1活性或表达的测试物质从而鉴定可用于在有此需要的受试者中抑制淋巴细胞增殖的测试物质。
25.权利要求24的方法,其中在这一具体实施方案中,所述筛选方法包含以下步骤:
a)在培养基中制备表达CTPS1的细胞的悬液,所述培养基用于支持所述细胞的代谢,
b)向所述悬液添加预定量的经标记的底物,
c)将步骤b)中获得的混合物在预定的温度孵育预定的一段时间,
d)从步骤中获得的所述孵育的混合物分离包含经标记的产物的部分,其通常通过裂解所述细胞以释放它们的细胞内含物,
e)检测步骤d)中获得的所述部分中经标记的产物的浓度,
f)在其他方面均相同的条件下添加预定量的所述测试物质来重复步骤b),c),d)和e),
g)通过观察步骤f)中检测到的经标记的产物的浓度是否比步骤e)中检测到的经标记的产物的浓度低确定对CTPS1的抑制的存在。
26.权利要求25的方法,其进一步包含由以下项组成的步骤:提供B或T细胞系,使得所述细胞系与选择的测试物质接触,确定所述B或T细胞系的增殖水平,将所述增殖水平与所述测试物质不存在时确定的增殖水平比较,和当所述测试物质存在时确定的增殖水平比所述测试物质不存在时确定的增殖水平低时,阳性选择所述测试物质。
27.权利要求25的方法,其包含其中在动物模型中测试所述测试物质的步骤。
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Also Published As
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US20180185476A1 (en) | 2018-07-05 |
US20160051674A1 (en) | 2016-02-25 |
JP2016523818A (ja) | 2016-08-12 |
WO2014170435A2 (en) | 2014-10-23 |
EP2986287A2 (en) | 2016-02-24 |
CA2909434A1 (en) | 2014-10-23 |
WO2014170435A8 (en) | 2014-12-11 |
WO2014170435A3 (en) | 2015-02-19 |
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