CN111902423A - 基于b7h受体的配体的抗肿瘤治疗剂 - Google Patents
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Abstract
包含至少一种B7h受体的配体的新型抗肿瘤剂,其中所述受体B7h的配体被装载到生物相容性的微米载体或纳米载体中,并且能够结合至受体B7h并触发受体B7h的活性。
Description
技术领域
本公开涉及包含至少一种受体B7h的配体的新型抗肿瘤治疗剂。
背景技术
ICOS是本发明人描述为H4的T细胞共刺激分子1,2。后来,Hutloff将ICOS克隆为属于CD28家族的分子,结果表明ICOS和H4是同一分子3,4。ICOS的显著特征是其在激活的T细胞中选择性表达,但最近在树突状细胞(DC)中也被检测到5。ICOS受体是ICOSL(或B7h),由多种细胞类型表达,如DC、巨噬细胞、B细胞、内皮细胞(EC)、上皮细胞和成纤维细胞6。ICOS/B7h相互作用调节淋巴器官中的T细胞激活,并控制炎症部位的T细胞功能。其支持Treg、TH17和滤泡辅助性T细胞的分化以及生发中心的发育,其缺陷会导致常见的可变免疫缺陷7-11。
B7h的其他名称是诱导性T细胞共刺激配体(ICOSL或ICOS-L),B7相关蛋白1(B7RP-1或B7RP1)B7同源物2(B7-H2),B7样蛋白Gl50、GL50、KIAA0653、LICOS、CD275。B7h:ICOS相互作用触发双向信号,调节表达B7h的细胞的应答。在小鼠DC中,这种B7h介导的“反向信号传导”增加了IL-6分泌12。为评估B7h在体外和体内触发的作用,本发明人产生了由IgG1的Fc部分和两个分子ICOS的胞外部分组成的ICOS的二价可溶性形式(ICOS-Fc)。由于ICOS-Fc是二价的,其与B7h交联并发挥激动作用,触发表达B7h的细胞中的B7h信号传导。在人细胞中,显示了由ICOS-Fc触发的B7h的下述作用。i)在EC和肿瘤细胞系中,其在体外抑制粘附和迁移13,14。ii)在肿瘤细胞系中,其抑制体内实验性肺转移的发展14。iii)在DC中,其通过增加IL-23的分泌来调节细胞因子的分泌(支持在抗肿瘤应答中涉及的TH17分化),促进抗原的交叉呈递,并且在体外抑制粘附和迁移15。iv)在破骨细胞(OC)中,其在体外抑制单核细胞的分化和骨吸收能力,并在体内抑制小鼠骨质疏松的发展16。v)B7h触发诱导EC中ERK和p38的去磷酸化,肿瘤细胞中FAK的去磷酸化以及在DC和肿瘤细胞中β-Pix的下调13-15。
发明内容
本公开的目的是提供包含至少一种受体B7h的配体的新型抗肿瘤治疗剂,其中所述受体B7h的配体能够特异性结合至受体B7h和触发受体B7h的活性。
根据本发明,由于所附权利要求中指定的方法实现了上述目的,所附权利要求被理解为构成本公开的组成部分。
本发明提供了一种受体B7h的配体,其用于治疗患有肿瘤的受试者,其中所述受体B7h的配体被装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上,并且所述受体B7h的配体能够结合所述受体B7h并触发所述受体B7h的活性。
附图说明
现在将参考附图,仅通过说明性和非限制性实例来详细描述本发明,其中:
图1:ICOS-Fc的不同形式对在C57BL/6小鼠中B16-F10肿瘤生长的影响。每4天给予具有皮下可触及B16肿瘤的小鼠PBS、单独的CDNS(纳米颗粒)、ICOS-Fc或装载ICOS-Fc的CDNS。在第1次治疗后16天评价肿瘤生长情况。每种治疗涉及5只小鼠/实验。**p<0.05vs每种其他条件。
图2:CDNS/ICOS-Fc对体内肿瘤血管生成的影响。对给予PBS、单独的CDNS或装载ICOS-Fc的CDNS的小鼠肿瘤组织切片,使用抗-CD31进行免疫荧光染色。使用Ab兔α-小鼠CD31以及二抗与Alexa488缀合的α-兔将玻片染色。显示了3个独立实验的代表性图像。条形图以肿瘤微血管密度(TDM)显示了这些实验的累积结果。**p<0.05vs每种其他条件。
图3:CDNS/ICOS-Fc对离体IL-10和Foxp3的影响。收集来自肿瘤的浸润细胞并用于实时PCR分析;针对在对照小鼠中的表达对数据进行归一化(将PBS组的对照表达设定为100%;*p<0.05)。
图4:ICOS-Fc的不同形式对在ICOS缺陷的C57BL/6小鼠中B16-F10肿瘤生长的影响。每4天给予具有皮下可触及B16肿瘤的小鼠单独的CDNS或装载ICOS-Fc的CDNS。(A)在第一次治疗后8天评价肿瘤生长;每种治疗涉及8只小鼠。**p<0.05。(B)通过实时PCR分析分析在肿瘤浸润淋巴细胞中IL-10和Foxp3的表达;针对在CDNS对照中的表达对数据进行归一化。从给予CDNS的小鼠的相应值得出#p<0.05。
图5:ICOS-Fc的不同形式对体外B16-F10细胞活力的影响。在存在和不存在滴定量(0.5-5μg/ml)的游离ICOS-Fc或空CDNS或CDNS/ICOS-Fc时,培养后,通过MTT评估的B16-F10细胞的细胞活力。结果显示为在不存在这些试剂的情况下培养的细胞中检测到的活力的抑制%。
图6:检测OPN与B7h相互作用的两种方法的结果。(左图)显示了滴定量的可溶性B7h-Fc(灰色线)与在ELISA板上包被的固定量的骨桥蛋白(OPN)的相互作用。黑线显示了在存在5μg/ml可溶性ICOS-Fc条件下进行的相同实验,以评价ICOS和OPN之间对B7h结合的竞争。虚线显示了缺乏滴定量的可溶性ICOS-Fc与包被OPN的板的结合。(右图)Western印迹显示了下拉测定,其中将B7h-Fc用作与OPN孵育(第一道)或未孵育(第二道)的琼脂糖结合的诱饵蛋白。第三道是OPN阳性对照。使用抗-OPN多克隆抗体对膜进行印迹。
图7:B7h在OPN功能中的作用。(A-B)通过OPN或FCS在表达高(A)和低(B)水平B7h的肿瘤细胞系中诱导的细胞迁移;**与未处理细胞的显著性差异。(C)在B7h转染(B7h高)的A2058细胞中恢复了对OPN的迁移应答;**与未转染细胞(B7h低)的显著性差异。(D)在B7h沉默(B7h低)的HUVEC中抑制了对OPN的迁移应答;**与未沉默细胞(B7h高)的显著性差异。水平虚线对应于未处理细胞的基础迁移,设定为100%。
图8:ICOS-Fc在OPN诱导的小管生成以及肿瘤细胞迁移和粘附中的作用。(A)ICOS-Fc对由OPN或VEGF诱导的HUVEC小管生成的作用;NT:无OPN和VEGF条件下的基管形成。**与使用ICOS-Fc处理的相应细胞的显著性差异。(B-C)ICOS-Fc对两种表达高水平B7h的人黑色素瘤细胞系(即,M14和JR8)迁移(B)和粘附(C)至HUVEC的影响。**p<0.05,与未处理细胞相比;§§p<0.05vs OPN处理的细胞。
图9:PLGA/ICOS-Fc在体外对B16-F10细胞存活以及在C57BL/6小鼠中B16-F10肿瘤生长的影响。(A)在存在和不存在滴定量(0.5-5μg/ml)的游离ICOS-Fc或空PLGA NP或PLGA/ICOS-Fc NP时,培养后,通过MTT评估的B16-F10细胞的细胞活力。结果显示为在不存在这些试剂的情况下培养的细胞中检测到的活力的抑制%。(B)每4天给予具有皮下可触及B16肿瘤的小鼠PBS、单独的PLGA(纳米颗粒)、PLGA/ICOS-Fc。在第1次治疗后16天评价肿瘤生长情况。每种治疗涉及5只小鼠/实验。*或**p<0.05vs每种其他条件。
图10:PLGA/ICOS-Fc对将人胶质母细胞瘤细胞注入大脑的无胸腺小鼠存活的影响。植入后7天和每7天,对腹腔内给予单独的PLGA(空白)(100μl)或使用人ICOS-Fc装载的PLGA(100μl)的负荷MD13肿瘤的无胸腺小鼠进行Kaplan-Meier分析。使用装载ICOS-FC的PLGA处理显著延长了荷瘤小鼠的存活率(在免疫受损小鼠中,空白vs ICOS-Fc,所有组n=6,P=0.00591,对数秩检验)。
具体实施方式
在以下描述中,给出了很多具体细节以提供对实施方式的透彻理解。可以在没有一个或多个具体细节的情况下或利用其他方法、部件、材料等来实施这些实施方式。在其他情况下,没有详细示出或描述公知的结构、材料或操作,以避免模糊实施方式的方面。
本说明书对“一个实施方式”或“实施方式”的提及意味着结合该实施方式描述的特定特征、结构或特性被包括在至少一个实施方式中。因此,本说明书在各个地方出现的短语“在一个实施方式中”或“在实施方式中”并不一定都指的是相同的实施方式。此外,特定特征、结构或特性可以以任何合适的方式在一个或多个实施方式中组合。
本文提供的标题仅仅是为了方便,并不解释实施方式的范围或含义。
本公开涉及新的抗肿瘤治疗剂,其包含具有触发受体B7h活性的能力的至少一种受体B7h的配体。
根据一个实施方式,本发明提供了一种受体B7h的配体,其用于治疗患有肿瘤的受试者,其中所述受体B7h的配体被装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上,并且所述受体B7h的配体能够结合所述受体B7h并触发所述受体B7h的活性,以及任选地抑制骨桥蛋白的活性。
在一个优选的实施方式中,受体B7h的配体选自:
a)具有SEQ ID No.:1或其部分所示氨基酸序列的人ICOS蛋白;
b)具有SEQ ID No.:2或其部分所示氨基酸序列的人ICOS胞外域;和
c)与SEQ ID No.:1、2或其部分所示氨基酸序列具有至少80%、优选地至少90%、更优选地至少95%、甚至更优选地至少98%的序列同一性的蛋白a)和b)中任一者的同源物。
在一个另外进一步优选的实施方式中,所述受体B7h的配体包含SEQ ID No.:3所示氨基酸序列。
在本发明的一个实施方式中,装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上的所述受体B7h的配体是通过注射、输注施用的。
如在本文中所使用的,表述“受体B7h的配体”包括具有SEQ ID No.:1所示氨基酸序列以及其部分(例如,具有SEQ ID No.:2所示的氨基酸序列)的人野生型配体ICOS,条件是这些部分具有结合受体B7h、触发其活性和任选地抑制骨桥蛋白活性的能力。
在本发明中使用的其他受体B7h的配体包括能够特异性结合受体B7h(即,具有高亲和性)的单克隆抗体,条件是这些单克隆抗体具有对B7h受体的激动活性,即其能够触发受体B7h的活性,和任选地具有对骨桥蛋白的拮抗活性,即其能够抑制骨桥蛋白的活性。
表述“人ICOS或其部分的同源物”指与SEQ ID No.:1和2的任一者或其部分具有至少80%、优选地至少90%、更优选地至少95%、甚至更优选地至少98%的同一性的蛋白,条件是这些同源物具有结合受体B7h和触发其活性,以及任选地抑制骨桥蛋白活性的能力。
通常的常识是,触发受体活性的能力意味着交联在细胞表面上表达的感兴趣的受体的能力,如在例如Seed B.“Making agonists of antagonists”,Chem Biol.1994Nov;1(3):125-9,和A,Schlessinger J.“Signal transduction by receptors with tyrosinekinase activity”,Cell.1990Apr 20;61(2):203-12中所示的。从这样的常识可以得出,受体B7h的配体必须至少为二聚体形式以发挥抗肿瘤作用。至少在能够触发受体B7h活性的二聚体形式中的配体的实例是例如二价或多价ICOS-Fc构建体、抗-B7h受体抗体、包含至少两个与其连接的ICOS分子的有机/无机的天然/合成支架以及由化学或遗传交联多个ICOS分子获得的ICOS多聚体。多聚化可以通过基于金属离子或小分子的组装、基于蛋白(肽)相互作用的组装、共价蛋白组装、功能性蛋白与蛋白构建嵌段的基因融合来实现。多聚化可以通过使用树枝状大分子(dendrimer)、树状物(dendron)、树枝状聚合物(dendronizedpolymers)、超支化聚合物和聚合物刷来实现。支架可以包括有机聚合物(如衍生自蔗糖、角鲨烯或茄尼醇的那些),以及在其表面上暴露多个ICOS分子的有机或无机固体纳米/微米颗粒。
在一个实施方式中,本发明提供了融合至或缀合至稳定分子的受体B7h的配体。
关于能够与受体B7h的配体缀合的稳定分子,此类分子在本领域中是众所周知的,因此不需要在本文中进行详细描述。作为稳定分子的实例,可以列举人Fc抗体结构域、聚乙二醇(PEG)或其衍生物、聚-L-赖氨酸柠檬酰胺(通过赖氨酸或氨基甲酸乙酯间隔基)、苯乙烯马来酸酐和聚羟丙基甲基丙烯酰胺。
能够连接在受体B7h的配体上存在的氨基的PEG衍生物是例如环氧PEG、醛PEG、碳酸硝基苯酯PEG和琥珀酰亚胺酯PEG;能够连接在受体B7h的配体上存在的巯基的PEG衍生物是例如邻吡啶基二硫化物PEG;能够连接在受体B7h的配体上存在的羟基的PEG衍生物是例如用N-羟基琥珀酰亚胺或羟基苯并***活化的PEG-COOH。其他PEG衍生物的代表是具有pH依赖性水解作用的PEG-聚缩醛和PEG-糊精(聚合物掩蔽-非掩蔽蛋白疗法,PUMPT)。
为了增加B7h受体的配体向治疗部位(即,肿瘤)的递送,受体B7h配体还可以被高糖基化或缀合至甘露糖残基。
可以通过原位化学反应或位点定向诱变进行高糖基化,从而导致N-连接或O-连接蛋白糖基化。在N-连接的糖基化中,糖链附接至三肽序列Asn-X-Ser/Thr的天冬酰胺上,其中X代表脯氨酸以外的氨基酸。通常将聚唾液酸(PSAlm)用于高糖基化。大分子量PSA适于递送低分子量药物和肽,而低分子量PSA能够用于较大蛋白和颗粒药物递送***。
与甘露糖残基的缀合利用了缀合物与甘露糖受体的结合,据报道该受体在枯否(Kupffer)细胞、巨噬细胞、肺泡、单核细胞衍生的树突状细胞以及血管和淋巴内皮细胞的亚群上表达。甘露糖基化蛋白可以被甘露糖特异性凝集素(即,甘露糖受体和MBP)识别。
关于生物相容性载体(其可以具有不同程度的生物降解性),所述载体选自颗粒、胶囊、囊泡、气泡,其各自具有微米或纳米量级的尺寸。其他适宜的载体由纳米乳、纳米混悬液、纳米水凝胶、胶束、树状聚合物、量子点、脂质体和碳衍生物(例如,碳纳米管)所代表。微米、纳米颗粒可以由聚合物、金属(例如,金)、二氧化硅或具有脂质外壳的合成聚合物核制成。金属颗粒也可以是磁性的。所有这些载体在用于药物递送的药物领域中是众所周知的,因此不需要在本文中详细描述。受体B7h的配体/载体***的抗肿瘤活性与载体的特定类型无关,但是受体B7h的配体与纳米/微米载体的缔合产生了一种微米、纳米平台制剂,其中两种组分共同起作用。将B7h受体的配体装载到微米/纳米载体中或微米/纳米载体上对于其治疗效果和临床转化是至关重要的。受体B7h的载体和配体同样有助于抗肿瘤活性,其协同作用对于有效的抗癌疗法是必不可少的。此外,如下所述,B7h受体配体/载体***对于放大抗肿瘤活性是重要的。将受体B7h的配体掺入载体中产生活性分子的储库并使其全身暴露最小化,从而允许其累积并释放至肿瘤组织并且避免了非特异性递送。事实上,在微米/纳米载体中掺入受体B7h的配体能够开发利用被动和主动靶向策略来递送至癌细胞的位点特异性靶向***。
在一个优选的实施方式中,载体由微米颗粒或纳米颗粒所代表,所述微米或纳米颗粒由环糊精聚合物、聚乳酸-羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、聚乳酸(PLA)、聚乙交酯、壳聚糖、藻酸盐、淀粉、藻酸盐、胶原、白蛋白、二氧化硅、金属制成。优选地,载体由微米颗粒或纳米颗粒所代表,所述微米或纳米颗粒由环糊精聚合物或聚乳酸-羟基乙酸共聚物制成。
在一个进一步的实施方式中,本发明涉及一种用于***的药物组合物,其包含至少一种装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上的受体B7h的配体和药学上可接受的载剂。
在一个优选的实施方式中,在药物组合物中包含的至少一种受体B7h的配体选自:
a)具有SEQ ID No.:1或其部分所示氨基酸序列的人ICOS蛋白;
b)具有SEQ ID No.:2或其部分所示氨基酸序列的人ICOS胞外域;和
c)与SEQ ID No.:1、2或其部分所示氨基酸序列具有至少80%、优选地至少90%、更优选地至少95%、甚至更优选地至少98%的序列同一性的蛋白a)和b)中任一者的同源物。
在另外一个优选的实施方式中,在药物组合物中包含的至少一种受体B7h的配体包含SEQ ID No.:3所示氨基酸序列。
在一个优选的实施方式中,在药物组合物中包含的至少一种受体B7h的配体被融合或缀合至稳定分子。
本公开还涉及受体B7h作为筛选用于***的药物活性剂的靶标的用途,其中所述药物活性剂结合至受体B7h,触发受体B7h的活性并抑制骨桥蛋白的活性。
此前的体外研究表明,ICOS-Fc触发B7h抑制DC、EC和几种肿瘤细胞系的迁移和粘附。此外,体内研究表明,ICOS-Fc抑制注入尾静脉的B7h阳性肿瘤细胞的肺转移。通过在小鼠尾静脉中注射人B7h阳性肿瘤细胞,并使用小鼠和小鼠ICOS-Fc(其是物种特异性的)治疗这些小鼠,本发明人证明了ICOS-Fc介导的体内转移作用的抑制涉及对肿瘤细胞和宿主细胞两者的影响。在同系小鼠尾静脉中注射B16黑色素瘤细胞后,使用ICOS-Fc进行体内治疗可抑制实验性肺转移的发展。此外,使用在NOD-SCID-IL2Rγnull(NSG)小鼠尾静脉中注射人CF-PAC1细胞系的异种移植模型,本发明人发现对转移的最佳抑制作用需要同时使用人和小鼠ICOS-Fc治疗。这些数据表明,ICOS-Fc通过同时作用于人肿瘤细胞和小鼠环境发挥其对转移性播散的抑制作用14。
肿瘤生长实际上涉及几个不同过程,如肿瘤细胞增殖和凋亡、组织侵袭、粘附和迁移、血管生成、血管内渗和血管外渗。因此,对肿瘤细胞粘附、迁移、血管内渗和血管外渗起作用的抗转移药物(称为“转移抑制”剂)对肿瘤生长无效,现在人们对于“转移抑制”剂的潜在临床意义提出了很大质疑17,18。实际上,发明人此前的实验在体内13,14或体外均未检测到ICOS-Fc对已建立的原发性肿瘤生长的任何作用。在体内检测了ICOS-Fc的几个剂量和递送途径(静脉内、腹腔内和肿瘤周围注射)以及表达B7h的肿瘤的几个实验模型(可移植的B16黑色素瘤和PC3***癌以及Balb/neuT自发性乳腺癌)获得了这些阴性结果。在体外实验中,事实上,未检测到ICOS-Fc对表达B7h的几种肿瘤细胞系的增殖和凋亡的任何作用14。此外,在体外,ICOS-Fc未显示出对血管内皮细胞增殖和血管生成测定的任何作用14。总之,在此前的研究中检测到的ICOS-Fc的唯一抗肿瘤作用是抗转移作用,而未检测到对体内肿瘤生长以及体外肿瘤细胞增殖和凋亡以及血管生成的作用。
然后,本发明人检测了在生物相容性纳米颗粒中包封的ICOS-Fc的抗肿瘤活性。
使用可移植肿瘤的B16黑色素瘤模型,本发明人意外地发现,包封在环糊精聚合物纳米海绵(CDNS)中的ICOS-Fc显示出有效的抗肿瘤活性,其减少已建立肿瘤块的生长并减少其血管生成。这些作用在ICOS缺陷型小鼠中也可以检测到,这表明其不仅归因于ICOS-Fc对B7h与内源性ICOS之间相互作用的拮抗作用。该作用不取决于纳米颗粒的类型和肿瘤类型,因为使用在聚乳酸-羟基乙酸共聚物(PLGA)纳米颗粒中装载的人ICOS-Fc和在胶质母细胞瘤的异种移植模型中检测到了相似的抗肿瘤作用。
B7h参与抗肿瘤应答的另一个方面是本发明人发现B7h还结合骨桥蛋白(OPN),骨桥蛋白可以充当细胞外基质蛋白和可溶性细胞因子。OPN由多种细胞类型分泌,包括巨噬细胞、DC、成骨细胞和T细胞。其介导多种功能,如骨骼重塑、巨噬细胞应答、细胞迁移和粘附、炎症以及支持TH1和TH17细胞分化。在肿瘤生物学中,由肿瘤细胞和肿瘤微环境产生的OPN在促进肿瘤生长、迁移、转移性扩散和新生血管生成中起关键作用。OPN与几种受体(如整联蛋白和CD44)相互作用,并在N末端(OPN-N)和C末端(OPN-C)部分被凝血酶裂解,其与不同受体结合并发挥不同功能。与几种整联蛋白结合的RGD结构域位于OPN-N中,其在凝血酶裂解后暴露的针对α4β1整联蛋白的两个其他结合位点附近;CD44结合位点位于OPN-C中19。本发明人出乎意料地发现,OPN诱导的肿瘤细胞系和EC的迁移应答需要B7h表达,并且该作用被ICOS-Fc抑制。然而,发现OPN使用不同于与ICOS结合的B7h结合位点直接与B7h结合。这些数据描述了一个场景,其中由ICOS触发的B7h抑制了由几种趋化因子诱导的细胞迁移,而由OPN触发的B7h诱导了细胞迁移,其中ICOS发挥主导作用。因此,不受到任何具体理论的束缚,本发明人有理由相信,ICOS-Fc还通过抑制OPN的作用(促进肿瘤生长、迁移、转移性扩散和新生血管生成)来发挥其抗肿瘤作用。
关于用于递送用于***的B7h受体的配体的载体,本发明人证实了载体的性质对B7h受体的配体发挥其抗肿瘤活性的能力没有任何影响。
微米或纳米颗粒可以由环糊精(CD)聚合物纳米海绵(CDNS)、聚乳酸-羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、聚乳酸(PLA)、聚乙交酯、壳聚糖、藻酸盐、淀粉、葡聚糖、胶原、白蛋白、二氧化硅、金属(例如,金)制成。也可以使用PEG对脂质体进行表面修饰,以干扰网状内皮***的识别和摄取,以延长循环时间。此外,原位热敏水凝胶会响应于温度变化而经历溶胶-凝胶相变。
CD是包含6个至>100个葡萄糖单元的环状α-1,4-葡聚糖。其是由CD葡聚糖转移酶降解的淀粉的分子内糖基转移反应产生的天然产物。酶产物通常是分别包含6个、7个和8个葡萄糖单元的α、β、γ-CD的混合物。其在超分子化学中起着重要作用,因为其具有与多种客体分子进行分子包封的能力,并且在制药领域、生物医学科学和生物技术领域引起了广泛关注。其呈现出具有内部疏水腔和亲水外部位点的环(torus)形环结构。由于存在CD腔且具有结晶或无定形结构的交联网状纳米通道、呈球形和具有溶胀性质,NS能够形成纳米多孔不溶性纳米颗粒(NP)20-24。
环糊精-纳米海绵(CDNS)可以形成能够容纳各种疏水性分子的包合物。在水溶液中,疏水性CD腔被受到“弱力”的束缚(在能量上是不利的)的水分子所占据。由于内部空腔的尺寸,因而一个或两个疏水性客体分子可以被一个、两个或者甚至三个CD所截留。CDNS可以形成球形的晶体或无定形的多孔性不溶性NP。通过改变交联的类型和程度,可以容易地调整聚合物网的极性和尺寸。CDNS可以掺入不同类型的亲脂性或亲水性分子,并且可以通过在其表面上缀合各种配体来对其进行官能化以实现位点特异性靶向。其是安全且可生物降解的,对细胞培养物的毒性可忽略不计,并且在小鼠体内注射后耐受性良好。可以通过调节NS的结构来调控被截留分子的释放,以实现改善水溶性差的分子的水溶性,保护可降解物质并获得持续释放。
聚乳酸-羟基乙酸共聚物(PLGA)NP是最具特征性的NP,可以增加多种药物的效力和生物利用度,其用途已获得美国食品和药物管理局(FDA)批准用于多种治疗用途。对聚乳酸羟基乙酸的比率、分子量和晶体特征的调控可以从几天到一年延长降解速率和随后被包裹分子的释放。
在本说明书中,本发明人发现仅当将ICOS-Fc包裹在生物相容性纳米颗粒中时,其才显著抑制已建立的原发性肿瘤的生长,而游离ICOS-Fc则没有作用(图1)。使用CDNS或PLGA纳米颗粒作为药物载体,以及使用小鼠肿瘤模型(黑色素瘤)(图1)或人/小鼠肿瘤异种移植模型(胶质母细胞瘤)(图9),在强免疫原性外周肿瘤(黑色素瘤)和弱免疫原性CNS肿瘤(胶质母细胞瘤)中均检测到了这一点。此外,ICOS-Fc不仅对具有免疫能力的小鼠(黑色素瘤)有效(图1),而且对无胸腺小鼠(胶质母细胞瘤)(图9)和ICOS-KO小鼠(黑色素瘤)(图4)也有效。这些数据表明,不能将ICOS-Fc的作用归因于阻断B7h与在T细胞(或其他细胞类型)上表达的内源性ICOS之间的相互作用。使用抗-ICOS mAb的这种阻断作用已显示出抑制Treg细胞的发育,这种细胞抑制抗肿瘤免疫应答。
不希望受到任何理论的束缚,本发明人有理由相信,载体(纳米颗粒)中装载的ICOS-Fc在发挥其抗肿瘤活性方面可以归因于载体通过增强的通透性和滞留效应(EPR)引起的携带ICOS-Fc进入肿瘤块的能力。各种类型的载体通过EPR或其他机制来增加药物向肿瘤的递送,可以发挥相同的作用,如与生物分子缀合的载体可以增加对肿瘤的靶向性。此外,其他能够触发B7h的分子(如单克隆抗体)也可以发挥出与ICOS-Fc相同的作用。
B7h的抗肿瘤活性可以部分归因于ICOS-Fc对肿瘤血管生成的作用,如ICOS-Fc在体外(图8)和体内(图2)的抗血管生成活性所提示的。这一发现是新的,因为此前的数据未检测到ICOS-Fc发挥的任何抗血管生成作用14。此外,人ICOS-Fc在胶质母细胞瘤异种移植模型中的作用表明,ICOS-Fc对肿瘤细胞也具有直接作用,因为人ICOS-Fc不与宿主细胞表达的小鼠B7h相互作用。为了支持包封在NP中的ICOS-Fc对肿瘤细胞活力的直接作用,发明人发现,包封在CDNS中的高剂量ICOS-Fc在体外对B16-F10细胞产生细胞毒性作用,而空CDNS和游离ICOS-Fc无作用(图5)。一种可能性是该差异可能是由于NP增加ICOS-Fc细胞内化的能力,这将增加ICOS-Fc与细胞内受体B7h的相互作用。
一个关键点是,首次证明了B7h也与OPN结合(图6),OPN是参与肿瘤生长、迁移和转移的关键分子,并且B7h/OPN相互作用参与这些OPN活动(图7)。B7h/OPN相互作用涉及与ICOS/B7h相互作用不同的结合位点(图6),而ICOS-Fc对OPN介导的几种促肿瘤活性表现出很强的显性负(dominant negative)效应(图8)。这些数据证实了ICOS-Fc对肿瘤生长的抑制活性不限于阻断B7h与T细胞上表达的内源性ICOS之间的相互作用。
鉴于本发明人进行的实验,ICOS-Fc是抗肿瘤药,其能够用于单一疗法或与其他抗肿瘤疗法的组合疗法。ICOS-Fc通过触发B7h和抑制OPN活性发挥作用,这是对抑制内源性ICOS/B7h相互作用的作用的实质性的另外作用。
结果
CDNS/ICOS-Fc对体内肿瘤生长的作用。
每4天向负荷可触及的皮下B16-F10肿瘤的C57BL/6小鼠i.v.给予小鼠ICOS-Fc、或ICOS-Fc/CDNS或空CDNS(各自100μg),或者相同体积PBS作为对照,且每4天监测一次肿瘤生长。结果表明,在CDNS中装载的ICOS-Fc(CDNS/ICOS-Fc)基本上抑制小鼠中黑色素瘤的生长,而游离ICOS-Fc没有作用(图1)。
CDNS/ICOS-Fc对体内肿瘤血管生成的作用。
为评估CDNS/ICOS-Fc对肿瘤血管生成的作用,评价了在使用B16-F10细胞获得的肿瘤中CD31的表达。结果表明,与空白小鼠(PBS)或给予空CDNS的小鼠相比,给予CDNS/ICOS-Fc减少血管形成(图2)。
CDNS/ICOS-Fc对离体免疫应答的作用。
为了评估治疗是否调节免疫应答,从肿瘤中获得了浸润的淋巴细胞,并通过实时PCR评价了IL-17A和RORγt(标记TH17细胞),IL-10和Foxp3(标记Treg细胞)的mRNA水平,因为ICOS在Th17和Treg细胞分化中起关键作用。结果表明,与在对照小鼠中检测到的水平相比,给予CDNS/ICOS-Fc显著降低Foxp3和IL-10的表达,而游离ICOS-Fc和空ICOS则无作用。而相反的是,在RoRγt和IL17A的表达中未检测到显著差异(图3)。
CDNS/ICOS-Fc对体内肿瘤生长的作用不取决于内源性ICOS的存在。
为评估ICOS-Fc的抗肿瘤作用在何种程度上取决于对B7h和内源性ICOS之间相互作用的抑制,评价了CDSN/ICOS-Fc对在ICOS缺陷小鼠中B16肿瘤生长的作用。结果表明,ICOS-Fc在ICOS缺陷小鼠中有效抑制B16肿瘤的生长(图4A)。通过从组织中提取的mRNA的实时PCR分析检测到,IL-10和FoxP3的表达降低伴随着对肿瘤生长的影响(图4B)。这些数据表明,ICOS-Fc的作用取决于B7h的触发,而不取决于B7h与内源性ICOS结合的拮抗作用。
CDNS/ICOS-Fc对体外肿瘤细胞的细胞毒性作用。
通过在用或不用滴定量(0.5、1、2、5μg/ml)的CDNS/ICOS-Fc、或空CDNS、或游离ICOS-Fc孵育的B16-F10细胞上进行MTT测定评价了CDNS制剂和游离ICOS-Fc的细胞毒性。结果表明,在最高剂量下检测到细胞毒性(在2和5μg/ml下有30%的抑制作用),其仅由CDNS/ICOS-Fc引起,而非空CDNS或游离ICOS-F(图5)。
B7h不仅与ICOS结合还与OPN结合
本发明人使用两种方法来检测OPN与B7h结合的假设,其使用重组B7h-Fc和带有组氨酸标签的OPN或B7h(B7h-his)(图6)。A)ELISA。将OPN(或B7h-his)吸附在ELISA板上,然后用滴定量的B7h-Fc(或OPN)孵育1h。洗涤后,使用抗-IgG1 mAb(或多克隆抗-OPN Ab)评价结合。结果显示了B7h与OPN浓度依赖性结合。此外,我们发现,OPN/B7h的结合不受ICOS-Fc的抑制,这表明OPN和ICOS-Fc与B7h的不同位点结合。ICOS-Fc未显示出与OPN的任何结合。B)下拉测定。将B7h-Fc用作在琼脂糖-蛋白A上捕获的诱饵蛋白,并与OPN孵育1h。洗涤后,从树脂上洗脱蛋白,并使用抗-OPN多克隆抗体通过Western印迹分析。结果表明,OPN与B7h之间存在关联。
OPN对B7h的触发促进肿瘤细胞系和EC的迁移
OPN诱导表达高水平B7h(B7h高)的肿瘤细胞系的迁移,但不诱导表达低水平B7h(B7h低)的肿瘤细胞系的迁移,而当使用胎牛血清(FBS)诱导迁移时,没有发现差异(图7A-B)。在B7h低肿瘤细胞中,通过增强B7h表达的B7h转染恢复了对OPN的迁移应答,而在B7h高HUVEC中,其被shRNA介导的B7h沉默所抑制(图6C-D)。由于B7h表达的调节不影响肿瘤细胞中FBS和HUVEC中VEGF诱导的迁移,因此这种作用是特异性的。
ICOS-Fc显著抑制OPN诱导的迁移和小管生成
在HUVEC中,给予ICOS-Fc抑制由OPN诱导的小管生成,但是不抑制由VEGF诱导的小管生成(图8A)。
在表达高水平B7h的肿瘤细胞系中,给予ICOS-Fc抑制由OPN诱导的细胞迁移和粘附至HUVEC(图8B)。
PLGA/ICOS-Fc对体内肿瘤生长的作用。
通过在用滴定量(0.5、1、2、5μg/ml)的空PLGA或PLGA/ICOS-Fc NP孵育的B16-F10细胞上进行MTT测定评估了PLGA制剂的细胞毒性。PLGA/ICOS-Fc NP仅在最高剂量时会产生一些毒性,而空PLGA NP或游离ICOS-Fc则不会(图9A)。
每4天向负荷可触及的皮下B16-F10肿瘤的C57BL/6小鼠i.p.给予小鼠PLGA/ICOS-Fc或空PLGA(各自100μg),或者相同体积PBS作为对照,且每4天监测一次肿瘤生长。结果表明,与两种对照治疗相比,给予PLGA/ICOS-Fc有效抑制B16-F10肿瘤的生长(图9B)。
将人神经母细胞瘤细胞(MD13)植入脑后一周,向在脑中负荷人胶质母细胞瘤的无胸腺小鼠给予装载人ICOS-Fc的PLGA或空PLGA。给予PLGA/ICOS-Fc显著延长了荷瘤小鼠的中位存活期(图10)。与给予空PLGA的小鼠相比,给予PLGA/ICOS-Fc减少了肿瘤形成。PLGA/ICOS-Fc对体内肿瘤生长的作用并不取决于内源性ICOS+CD4+T细胞的存在,因为我们评价了PLGA/ICOS-Fc在T细胞缺陷小鼠中的作用。
材料和方法
ICOS和ICOS-Fc的克隆和生产
将人或小鼠ICOS的胞外部分克隆到衍生自pCDNA3.1/Hygro(+)质粒(商品编号:V870-20,Invitrogen)的经修饰的真核表达载体中,并由Di Niro R.等25报道为p-Minibody(pMB-SV5),PubMed ID:17678525。该载体与原始载体的不同之处在于:在真核细胞中启动翻译所需的Kozak序列(5’CCACCATGG 3’–SEQ ID No.:11);分泌前导序列(5’GCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGCTACA 3’–SEQ ID No.:12),引入其以使得在培养物上清液中释放蛋白;微型内含子序列(5’GGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATAT ATGGGTGACAATGACATCCTTTGCCTTTCTCTCCACAGGTG 3’-SEQ ID No.:13),以提高蛋白表达水平。引入了靶向所生产蛋白的标签序列,该标签序列衍生自猿猴病毒-5(SV5标签)(5’GGCAAACCAATCCCAAACCCACTGCTGGGCCTGGATAGTACT
3’-SEQ ID No.:14),其可用于对蛋白进行单克隆抗体识别。该载体能够使用人或小鼠免疫球蛋白IgG1(Fc)结构域的恒定片段的编码序列在框中克隆感兴趣的片段,所述编码序列分别具有SEQ ID No.:5和9所示的核苷酸序列。
为产生人ICOS-Fc构建体(SEQ ID No.:3),使用下述特异性引物扩增编码人ICOS胞外部分的核苷酸序列(SEQ ID No.:4):ICOS正向BsshII引物(5’TTGGCGCGCATGCCGAAATCAATGGTTCTGCC 3’–SEQ ID No.:15,Sigma-Genosys,The Woodlands,TX,USA)和ICOS反向NheI引物(5’CTAGCTAGCAAGTTGTGATTCATAAATATGC 3’-SEQ ID No.:16,Sigma-Genosys)。用BssHII(商品编号:R0199S,New England Biolabs inc,Ipswich,MA,USA)和NheI(商品编号:R0131S,New England Biolabs inc)酶对扩增的片段进行消化。将双重消化的片段克隆至具有人Fc结构域的编码序列(人Fc结构域具有SEQ ID No.:5所示的序列)的此前所述的pMB-SV5质粒。通过测序确定核苷酸序列。编码huICOS-huFc的表达载体的核苷酸序列如SEQID No.:6中所示。
为产生小鼠ICOS-Fc构建体(SEQ ID No.:7),使用下述特异性引物扩增编码小鼠ICOS胞外部分的核苷酸序列(SEQ ID No.:8):ICOS小鼠正向BsshII引物(5’TTGGCGCGCATGCCGAAATCAATGGCTCG 3’–SEQ ID No.:17,Sigma-Genosys)和ICOS小鼠反向NheI引物(5’CTAGCTAGCTAGCCAGAGCTTCAGCTGGC 3’–SEQ ID No.:18,Sigma-Genosys)。用BssHII(商品编号:R0199S,New England Biolabs inc,Ipswich,MA,USA)和NheI(商品编号:R0131S,New England Biolabs inc)酶对扩增的片段进行消化。在该克隆中使用的载体是具有小鼠Fc结构域的编码序列(小鼠Fc结构域具有SEQ ID No.:9所示的序列)的pMB-SV5。将双重消化的片段克隆至此前所述的pMB-SV5质粒。通过测序确定核苷酸序列。编码msICOS-msFc的表达载体的核苷酸序列如SEQ ID No.:10中所示。
将质粒DNA转化至OneTOP10化学感受态大肠杆菌细菌细胞(E.coli;商品编号:C4040-03,Life Technologies,Carlsbad,CA,USA)中。使用下述特异性引物筛选所得到的菌落:P-Hygro正义(5’CTGCTTACTGGCTTATCG 3’–SEQ ID No.:19,Sigma-Genosys)和P-Hygro反义(5’CAGATGGCTGGCAACTAG 3'–SEQ ID No.:20,Sigma-Genosys),并且通过测序确认了构建体。最后,使用FreeStyleTMMAX试剂(商品编号:16447100,Life technologies)将质粒DNA转染到中国仓鼠卵巢混悬细胞系(CHO-s)(商品编号:R8/00-07,Invitrogen)中。由于在载体中存在潮霉素抗性,因而获得了稳定的克隆;为此,使克隆在0.2mg/ml的浓度的潮霉素-B(商品编号:10687-010,Invitrogen)的选择下生长,以使得能够完全选择转染细胞。使细胞在无血清IMDM培养基(商品编号:BE12-915F01,Lonza,Basel,Switzerland)中生长,使用蛋白G SepharoseTM 4 Fast Flow柱(商品编号:17-0618-01,GE Healthcare,Piscataway Township,NJ,USA)纯化无血清培养物上清液。
CDNS和CDNS/ICOS-Fc的制备
如先前报道20,21,制备了与碳酸酯桥交联的含β-环糊精的碳酸酯NS(CDNS;代码C4767,Sigma-Aldrich,St.Luis,MO,USA)作为构建嵌段。简言之,将一定量的无水CD溶于无水二甲基甲酰胺(DMF;代码227056,Sigma-Aldrich,St.Luis,MO,USA)中,并使其与羰基二咪唑(代码115533,Sigma-Aldrich,St.Luis,MO,USA)在90℃下反应至少5h。一旦反应结束,就加入大量过量的水以破坏过量的羰基二咪唑(代码115533,Sigma-Aldrich,St.Luis,MO,USA),通过过滤回收固体并使用水纯化。然后,将固体在研钵中研磨,并用乙醇(代码51976,Sigma-Aldrich)进行索式提取,以除去残留的反应副产物。使用摩尔过量的交联剂(例如,1:4环糊精:交联剂)进行反应。纯化后,将干燥的NS保存在25℃。
将β-CDNS与苯四甲酸二酐(代码412287,Sigma-Aldrich,St.Luis,MO,USA)交联形成羧酸封端的纳米多孔材料(βNS-Pyro),其形成具有相当的球形和对难溶物质具有非常高的溶解能力的固体颗粒。为了获得βPyro-NS,将苯四甲酸二酐(代码412287,Sigma-Aldrich,St.Luis,MO,USA)加入无水环糊精中。在无水二甲亚砜(DMSO,代码D1435,Sigma-Aldrich,St.Luis,MO,USA)中,在室温下进行合成24h。在存在氨的条件下,通过以1:8的摩尔比率交联-CDNS和苯四甲酸二酐(代码412287,Sigma-Aldrich,St.Luis,MO,USA)来制备摩尔βNS-Pyro。对于生物学实验,使用Ultraturrax仪器(IKA,Germany),将干燥的制剂以10mg/ml浓度分散在0.9%NaCl(代码S7653,Sigma-Aldrich,St.Luis,MO,USA)溶液中3min。所有试剂均为分析纯。
通过拉曼光谱和成像方法考察NP的结构。通过透射电子显微镜的我们的数据表明,形成的NP具有几乎球形的形态,尺寸范围为50至100nm,平均81±9nm。该结果表明,NS-Pyro NP没有炎症作用,且对黑色素瘤细胞系没有任何不利影响。然后,发明人将ICOS-Fc包封进入这些βNS-Pyro NP中。通过搅拌将一定量的冻干βNS-Pyro分散在pH 6.0的含NaCl(代码S7653,Sigma-Aldrich,St.Luis,MO,USA)和PEG 400(聚乙二醇400,代码202398,SigmaAldrich,St.Luis,MO,USA)3%w/v的水溶液中,以获得含有ICOS-Fc的等渗水性纳米混悬液。
PLGA-纳米颗粒(NP)的生产
通过改进的双溶剂蒸发法制备PLGA纳米颗粒26。简言之,在室温下,将60mg PLGA65:35晶体(商品编号:P2066;Sigma-Aldrich,Saint Luis,MO,USA)溶解在1ml二氯甲烷(DCM)(商品编号:270997;Sigma-Aldrich,St.Luis,MO,USA)中。将50μl PBS加入PLGA中,并将溶液超声1min。将5体积1%PVA(商品编号:P8136;Sigma-Aldrich,St.Luis,MO,USA)的水溶液小心地添加到所得到的乳液中,以保持相分离。再超声2min以获得最终的乳液,将其在通风橱中蒸发过夜以除去DCM。通过以7,000rpm离心10min在蒸馏水中将所得到的纳米颗粒洗涤7次,并重悬于0.9%的NaCl中,并且在4℃下保存。通过在制备的第一步中,将ICOS-Fc(冻干1mg,然后重悬于50μl PBS磷酸盐缓冲液中)加入到溶解在DCM中的PLGA溶液中,按照上述方法制备出含有ICOS-Fc的纳米颗粒。通过在37℃下将纳米颗粒留在PBS中来评价ICOS-Fc的释放,并通过BCA测定(ThermoScientific)对释放的蛋白进行定量。
体内实验
皮下注射(s.c.)给予雌性5-7周龄C57BL/6小鼠(野生型,商品编号:000664-C57BL/6J或ICOS-/-商品编号:004859-B6.129P2-Icostm1Mak/J;Charles RiverLaboratories,Wilmington,MA,USA)B16-F10细胞(105个细胞/小鼠;商品编号:CRL-6475;ATTC,Manassas,VA,USA)。10天后,当肿瘤可触及时,每4天向小鼠静脉内(i.v.)注射给予小鼠ICOS-Fc、在CDNS中装载的ICOS-Fc(ICOS-Fc/CDNS)或空CDNS(各自100μg)或者相同体积PBS作为对照。用卡尺每4天测量一次肿瘤尺寸,并在3周后处死小鼠。在其他实验中,应用相同治疗方案给予小鼠在PLGA中装载的ICOS-Fc(ICOS-Fc/PLGA)或空PLGA NP(各自100μg)或相同体积PBS作为对照。
在其他实验中,向无胸腺小鼠(Balb/c nu/nu;品系代号194,Charles-River)的右纹状体立体定向注入从刚切除的神经胶质瘤肿瘤细胞培养物中获得的1x 104个人类分离的胶质母细胞瘤肿瘤球细胞(间质表型)27,其对应于干细胞样肿瘤细胞,表达B7h(每个治疗组n=6)。肿瘤攻击7天后,腹腔注射给予小鼠100μl含ICOS-Fc的PLGA纳米颗粒或空PLGA。当在脑荷瘤动物中出现神经病理学体征时,处死小鼠。为了获得肿瘤球细胞,将新鲜切除的神经胶质瘤样品解离,并在特定培养基中培养建立的细胞,所述培养基含有补充了B27(此前称为MACS Supplement B27 PLUS,商品编号:130-093-566Miltenyi Biotec,Bergisch Gladbach,Germany)和肝素(2.5μg/ml,商品编号:H3149,Sigma-Aldrich,St.Loius,MO,USA)的DMEM/F12(商品编号:31331-028,Thermo Fisher,Waltham,USA)。为增强增殖和保持干性,每周2次向球形培养物中添加碱性成纤维细胞生长因子(bFGF;20ng/ml,商品编号:100-18B,Peprotech,London,UK)和表皮生长因子(EGF;20ng/ml,商品编号:AF-100-15Peprotech,London.UK)27。在无病原体条件下,在健康科学系的动物房中饲养小鼠,并根据大学伦理委员会的要求对其进行处理。本研究得到了皮埃蒙特东方大学动物实验生命伦理委员会和***(the Bioethics Committee for AnimalExperimentation of the University of Piemonte Orientale and Ministero dellaSalute)的批准(方案号:477/2016-PR)。
实时逆转录聚合酶链反应
从肿瘤获得浸润细胞,并通过实时PCR评价IL-17A和RORγt(标记TH17细胞)、IL-10和Foxp3(标记Treg细胞)的mRNA水平。然后,使用TRIzol试剂(商品编号:15596026,Thermo Fisher,Waltham,USA)分离总RNA。使用QuantiTect逆转录试剂盒(商品编号:205311,Qiagen,Hilden,Germany)对RNA进行逆转录。使用基因表达测定(商品编号:4453320,Assay-on Demand,Applied Biosystems,Forest City,CA,USA)评价其表达。使用GUSB基因对cDNA的量进行标准化。使用CFX96***(Bio-Rad Laboratories)进行实时PCR,一式两份,每个样品终体积10μl,其中含1μl稀释的cDNA、5μl TaqMan通用PCR预混液(商品编号:4369016Applied Biosystem,Foster City,CA)和0.5μl TaqMan基因表达测定(Applied Biosystem)。使用了下述测定:GUSB,Mm01197698_m1;IL-17A,Mm00439618_m1;RORγt,Mm01261022_m1;IL-10,Mm01288386_m1;Foxp3,Mm00475162_m1。热循环仪的参数是95℃下10min,然后是40个循环的95℃下15sec和60℃下1min。使用Delta-Delta CT法分析结果。
抗-CD31免疫荧光
解剖后,立即将肿瘤样品包埋在OCT化合物(商品编号:05-9801,Killik,BiopticaMilano SpA)中,速冻,并于-80℃下储存直至使用。用冷冻切片机切取肿瘤组织(厚度为5-6μm),并且在室温下用在PBS中稀释的4%多聚甲醛(商品编号:P6148,Sigma-Aldrich)处理5分钟,以便将样品固定在载玻片上。然后,使用含5%正常山羊血清(NGS cod.DY005 R&DSystem,Minneapolis,USA)的PBS封闭样品1小时,以封闭能够与一抗结合的非特异性位点。为检测CD31的表达,使用一抗兔抗-CD31(商品编号:ab28364,1:50稀释,Abcam,Cambridge,UK)将切片在室温下孵育2小时。使用的二抗是与Alexa fluor 488缀合的抗-兔Ig(商品编号:A-11008,Thermo Fisher),1:400稀释。然后,将切片用0.5mg/ml的荧光染料4,6-二脒基-2-苯基吲哚-二盐酸盐(DAPI,商品编号:D8417,Sigma-Aldrich)染色5分钟,以使细胞核着色,然后使用Prolong防褪色封片剂(Slow Fade AntiFADE试剂盒,商品编号:S2828,Molecular Probes Invitrogen)封片。随后,使用荧光显微镜(Leica,Italy)观察切片,并通过用于显微成像的Image Pro Plus软件5.0(Media Cybernetics,5.0版,Bethesda,MD,USA)进行分析。
细胞迁移测定
在Boyden小室(BD Biosciences,San Diego,CA,USA)迁移测定中,将黑色素瘤细胞(A2058,商品编号:CRL-11147,ATCC,Manassas,VA,USA;M14,RRID:CVCL_1395;JR8,RRID:CVCL_5780;PCF-2)接种在50μg/ml matrigel包被滤器(商品编号:直径8.2mm,孔径0.3μm或0.5μm;Neuro Probe,Inc.;BIOMAP snc,Milan,Italy;Matrigel基质基底膜,商品编号:L003975Rif.Cat 354230,SACCO s.r.l.,COMO,Italy)的顶侧,在含有或不含OPN(10μg/ml,商品编号:1433-OP-050,R&D System,Minneapolis,USA)或ICOS-Fc(5μg/ml)的RPMI-1640(代码BE12-702F/12,Lonza,Basel,Switzerland)无血清培养基中。将含20%FCS(商品编号:10270106,Gibco,Gaithersburg,MD,USA)的培养基作为正向趋化因子刺激置于基底外侧室中。将小室在37℃下5%CO2中孵育。20h后,使用Q-tip棉签抹去顶侧上的细胞。滤器底部的细胞用结晶紫(商品编号:61135,Sigma-Aldrich,St.Louis,MO,USA)染色,并全部用倒置显微镜(40倍放大)计数(四倍滤器)。数据以经处理细胞迁移相对于未经处理细胞测得的对照迁移的百分比表示。
细胞粘附测定
使HUVEC在24孔板(商品编号:ET3024,Euroclone,Milan,Italy)的完全培养基M200(商品编号:M200500,Gibco,Gaithersburg,MD,USA)中生长至汇合,然后使用或不使用OPN(10μg/ml,商品编号:1433-OP-050,R&D System,Minneapolis,USA)或ICOS-Fc(5μg/ml)处理30min,使用新鲜培养基洗涤两次,并与黑色素瘤细胞(5x104个/孔;A2058,商品编号:CRL-11147,ATCC,Manassas,VA,USA;M14,RRID:CVCL_1395;JR8,RRID:CVCL_5780;PCF-2)孵育1h。在粘附测定中孵育后,通过用M200培养基洗涤3次除去未粘附细胞。通过荧光成像分析来分析每个孔的中心。通过用于显微成像的Image Pro Plus软件(Media Cybernetics,Bethesda,MD,5.0版)计数粘附细胞。数据以经处理细胞的粘附相对于未经处理细胞测得的对照粘附的百分比表示。
小管形成测定
在小管形成测定中28,将HUVEC(2.5x104/孔)在M200(商品编号:M200500,Gibco,Gaithersburg,MD,USA)无血清培养基中培养,并且在存在OPN(10μg/ml,商品编号:1433-OP-050,R&D System,Minneapolis,USA)的条件下或含VEGF-α(10ng/ml,商品编号:293-VE-010;R&D System,Minneapolis USA)的对照培养基中,将细胞接种在预先使用150μl生长因子减少的matrigel(Matrigel基质基底膜,商品编号:L003975Rif.Cat 354230,SACCOs.r.l.,COMO)包被的48孔板(商品编号:ET3048,Euroclone,Milan,Italy)上。培养6h后,使用倒置显微镜(Leica Microsystem,Milano,Italy;放大倍数10X)分析由HUVEC形成的毛细管样结构的形态,并用数码相机(Leica Microsystem,Milano,Italy)照相。分析小管形成情况并用成像***(用于显微成像的Image-Pro Plus软件,Media Cybernetics,5.0版,Bethesda,MD,USA)计数小管数量(在两端具有分支)。通过计数在3个孔中的总小管数评价小管形成情况(n=5)。
下拉测定
将10ug rhB7h2-Fc(商品编号:165-B7-100,R&D system,Minneapolis,MN,USA)和rhOPN(商品编号:1433-OP-050/CF,R&D system,Minneapolis,MN,USA)一起加入PBS中,在RT下在转子上离心1h,然后使用琼脂糖-蛋白G(商品编号:17-0618-01,GE Healthcare,Piscataway,NJ,USA)沉淀B7h,将含20%β-巯基乙醇(商品编号:M-3148,Sigma-Aldrich,Saint Louis,MO,USA)的样品缓冲液用于解离蛋白,并进行Western印迹。将抗-OPN多克隆抗体(商品编号:MAB14331-SP,R&D system,Minneapolis,MN,USA)用于检测膜上的OPN。
B7h沉默和转染
对于B7h沉默实验,将HUVEC细胞(1.5*10^5个细胞)接种在6孔板(商品编号:ET3006,Euroclone,Milan,Italy)中的完全培养基IMDM(商品编号:BE12-915F01,Lonza,Basel,Switzerland)中24h。为沉默细胞,将lipofectaminetm RNAiMAX转染试剂(商品编号:13778030,Life technologies,Carlsbad,CA,USA)与映射在两个不同外显子中的两个不同siRNA导向(direct)B7h(oligo1:ICOSLGHSS177318(5’-CAGCAGCCUUCGAGCUGAUACUCAG-3’–SEQ ID No.:21和5’-CUGAGUAUCAGCUCGAAGGCUGCUG-3’–SEQ ID No.:22)和oligo2:ICOSLGHSS118565(5’-GGCCCAACGUGUACUGGAUCAAUAA-3’–SEQ ID No.:23和5’-UUAUUGAUCCAGUACACGUUGGGCC-3’–SEQ ID No.:24),Life technologies,Carlsbad,CA,USA)一起使用。对于B7h转染实验,将A2058细胞(10^6个细胞;商品编号:CRL-11147,ATCC,Manassas,VA,USA)接种在10cm2培养皿(商品编号:ET2100,Euroclone,Milan,Italy)中的完全培养基RPMI-1640(代码:BE12-702F/12,Lonza,Basel,Switzerland)中。为转染细胞,使用10μg DNA和10μl lipofectamine3000(代码:L3000001,Life technologies,Carlsbad,California,USA)。24或48小时后,将沉默或转染的细胞用于迁移实验。
ELISA测定
将80nM rhOPN(#1433-OP-050/CF,R&D system,Minneapolis,MN,USA)吸附在NuncMaxiSorpTM平底ELISA板(商品编号:M9410-1CS,Sigma-Aldrich,St.Louis,MO,USA)上,然后在存在或不存在80nM rhICOS-Fc的条件下,与滴定量B7h-Fc(商品编号:165-B7-100,R&Dsystem,Minneapolis,MN,USA)孵育1h。使用PBS+0,025%Tryton(商品编号:T8787,Sigma-Aldrich,St.Louis,MO,USA)洗涤后,加入HRP缀合的抗-人IgG1 mAb(商品编号:P0214,Dako,Santa Clara,CA,USA)作用1h,然后加入TMB底物(商品编号:T4444,Sigma-Aldrich,St.Louis,MO,USA)并使用H2SO4 2N(商品编号:339741,Sigma-Aldrich,St.Louis,MO,USA)作用2min终止反应,使用Victor-X1酶标仪(Perkin Elmer,Waltham,MA,USA)在450nm下读取吸光度。
细胞活力测定
以1x103个细胞/孔在RPMI-1640完全培养基中,将B16-F10细胞接种在96孔板中。24h后,除去培养基,将细胞在含滴定量(0.5-5μg/ml)CDNS或PLGA NP的培养基中孵育48h。孵育72h后,通过添加2,3-双[2-甲氧基-4-硝基-5-磺苯基]-2H-四唑-5-甲酰胺(MTT,Sigma-Aldrich)内盐试剂(0.5mg/ml)在37℃下作用4h以评价活细胞。然后,弃去MTT溶液,并用100μl DMSO(Sigma-Aldrich)溶解甲臜结晶。在微孔板分光光度计(Perkin Elmer,Waltham,Massachusetts,USA)中,在570nm处测量吸光度。使用下述公式计算细胞活力:细胞活力=样品的吸光度/对照的吸光度x 100(n=5)。
数据分析
使用GraphPad Instat软件(GraphPad Software,San Diego,CA,USA),通过Mann-Whitney检验进行统计学分析。数据以平均值±SEM表示,将统计学显著性设定为p<0.05(Mann-Whitney检验)。
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Claims (15)
1.受体B7h的配体,其用于治疗患有肿瘤的受试者,其中所述受体B7h的配体被装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上,并且所述受体B7h的配体能够结合所述受体B7h并触发所述受体B7h的活性。
2.根据权利要求1所述的受体B7h的配体,其中所述配体选自:
a)具有SEQ ID No.:1或其部分所示氨基酸序列的人ICOS蛋白;
b)具有SEQ ID No.:2或其部分所示氨基酸序列的人ICOS胞外域;和
c)与SEQ ID No.:1、2或其部分所示氨基酸序列具有至少80%、优选地至少90%、更优选地至少95%、甚至更优选地至少98%的序列同一性的蛋白a)和b)中任一者的同源物。
3.根据权利要求1或权利要求2所述的受体B7h的配体,其中所述配体被高糖基化或缀合至甘露糖残基。
4.根据权利要求1至3中任一项所述的受体B7h的配体,其中装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上的所述配体是通过注射、输注施用的。
5.根据权利要求1至4中任一项所述的受体B7h的配体,其中所述配体被融合或缀合至稳定分子。
6.根据权利要求5所述的受体B7h的配体,其中所述稳定分子选自:人Fc抗体结构域、聚乙二醇及其衍生物、聚-L-赖氨酸柠檬酰胺、苯乙烯马来酸酐和聚羟丙基甲基丙烯酰胺。
7.根据权利要求1至6中任一项所述的受体B7h的配体,其中所述配体包含SEQ ID No.:3所示氨基酸序列。
8.根据权利要求1至7中任一项所述的受体B7h的配体,其中所述生物相容性的微米载体或纳米载体选自:微米或纳米颗粒、微米或纳米胶囊、微米或纳米囊泡、微米或纳米气泡、纳米乳、纳米混悬液、纳米水凝胶、胶束、树状聚合物、量子点、脂质体和碳衍生物。
9.根据权利要求8所述的受体B7h的配体,其中所述微米或纳米颗粒由环糊精聚合物、聚乳酸-羟基乙酸共聚物、聚己内酯(PCL)、聚乳酸(PLA)、聚乙交酯、壳聚糖、藻酸盐、淀粉、藻酸盐、胶原、白蛋白、二氧化硅、金属制成。
10.用于***的药物组合物,其包含至少一种装载到生物相容性的微米载体或纳米载体中或生物相容性的微米载体或纳米载体上的受体B7h的配体和药学上可接受的载剂。
11.根据权利要求10所述的药物组合物,其中所述至少一种受体B7h的配体选自:
a)具有SEQ ID No.:1或其部分所示氨基酸序列的人ICOS蛋白;
b)具有SEQ ID No.:2或其部分所示氨基酸序列的人ICOS胞外域;和
c)与SEQ ID No.:1、2或其部分所示氨基酸序列具有至少80%、优选地至少90%、更优选地至少95%、甚至更优选地至少98%的序列同一性的蛋白a)和b)的任一者的同源物。
12.根据权利要求10或权利要求11所述的药物组合物,其中所述配体被融合或缀合至稳定分子。
13.根据权利要求10至12中任一项所述的药物组合物,其中所述配体包含SEQ ID No.:3所示氨基酸序列。
14.根据权利要求10至13中任一项所述的药物组合物,其中所述生物相容性的微米载体或纳米载体选自:微米或纳米颗粒、微米或纳米胶囊、微米或纳米囊泡、微米或纳米气泡、纳米乳、纳米混悬液、纳米水凝胶、胶束、树状聚合物、量子点、脂质体和碳衍生物。
15.受体B7h作为筛选用于***的药物活性剂的靶标的用途,其中所述药物活性剂结合至受体B7h,触发受体B7h的活性并抑制骨桥蛋白的活性。
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IT201800001315A1 (it) | 2019-07-18 |
US20210128684A1 (en) | 2021-05-06 |
WO2019142070A1 (en) | 2019-07-25 |
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