CN111556758A - 细胞杀伤剂 - Google Patents
细胞杀伤剂 Download PDFInfo
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- CN111556758A CN111556758A CN201880085530.7A CN201880085530A CN111556758A CN 111556758 A CN111556758 A CN 111556758A CN 201880085530 A CN201880085530 A CN 201880085530A CN 111556758 A CN111556758 A CN 111556758A
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Abstract
本发明的课题在于,提供一种肽药剂,其可针对靶细胞选择性地诱导细胞凋亡。本发明涉及由SEQ ID NO:1所表示的氨基酸序列组成的、具有内涵体逃逸肽与细胞凋亡诱导肽的融合肽的细胞杀伤剂;和上述记载的细胞杀伤剂,其为由细胞异常增殖引起的疾病的治疗剂。
Description
技术领域
本发明涉及可针对靶细胞选择性地诱导细胞凋亡的肽药剂。
本申请基于2018年1月9日在日本申请的特愿2018-001063号要求优先权,并将其内容援引于此。
背景技术
药品的有效成分根据其大小分为低分子药(低分子化合物药)、抗体药物、和大于低分子化合物且小于抗体的中分子药。在中分子药中,例如包含肽药剂。低分子药、中分子药、抗体药物分别具有优点或问题。例如,低分子药由于分子量较小,因此可穿过细胞膜并进入靶细胞的内部,但无法阻碍蛋白质之间的结合。另外,尽管具有制造成本相对廉价的优点,但筛选已经进行了很长时间,并且新型分子的搜索处于困难的状态。另一方面,抗体药物对靶分子的特异性或结合力高,而且可阻碍蛋白质之间的结合,但由于分子量较大而不能穿过细胞膜,无法进入靶细胞的内部。另外,制造成本高是较大的缺点。中分子药兼具低分子药和抗体药物的优点。即,除了对靶分子的特异性或结合力高之外,还可穿过细胞膜并作用于细胞内的靶分子。另外,由于通常可进行化学合成,因此制造成本较抗体药物低,且可合成纯度高的药剂。
另一方面,细胞凋亡为多细胞生物的细胞死亡中、作为増殖控制机制而被管理/调节的细胞死亡。在多细胞生物中,通过对这些细胞诱导细胞凋亡,来排除在细胞生成或再生过程中所产生的不必要的细胞或有害的细胞。另外,通过对引起疾病的细胞诱导细胞凋亡并进行排除,可期待病情的改善,因此也将具有诱导细胞凋亡的活性的物质用于药物用途。例如,可期待通过针对癌细胞诱导细胞凋亡,来缓解或治愈癌症。
作为对引起疾病的细胞诱导细胞凋亡的肽药剂,例如,报道了一种肽组合物,其包含与CNGB3(cyclic nucleotide-gated channel beta 3)特异性结合的Z13肽与内涵体逃逸肽的融合肽、以及Z13肽与细胞凋亡诱导肽的融合肽,所述CNGB3是在子宫内膜异位症细胞(存在于除子宫内膜以外的子宫内膜细胞)的细胞表面上特异性表达,而在腹膜表面不表达的分子(参见专利文献1)。通过Z13肽部分将两种肽一起摄入子宫内膜异位症细胞,由此可针对子宫内膜异位症细胞选择性地诱导细胞凋亡。实际上,当对患有子宫内膜异位症的狒狒,通过腹腔镜向腹膜给予包含Z13肽与内涵体逃逸肽的融合肽、以及Z13肽与细胞凋亡诱导肽的融合肽的肽组合物时,仅子宫内膜异位症病变的细胞被选择性地诱导细胞凋亡,而相邻的其他细胞则没有被诱导细胞凋亡(参见非专利文献1)。
现有技术文献
专利文献
专利文献1:美国专利申请公开第2016/145308号说明书;
非专利文献
非专利文献1:Sugihara等人,NATURE COMMUNICATIONS、2014年、第5卷、第4478号。
发明内容
发明所要解决的课题
本发明的目的在于,提供可针对靶细胞选择性地诱导细胞凋亡的肽药剂。
用于解决课题的手段
本发明人为了解决上述课题进行了深入研究,结果发现:由特定的肽序列组成的内涵体逃逸肽与由特定的肽序列组成的细胞凋亡诱导肽的融合肽,能够以较高的选择性、有效地针对癌细胞诱导细胞凋亡,从而完成了本发明。
即,本发明提供以下的细胞杀伤剂和由细胞异常增殖引起的疾病的治疗方法。
[1]细胞杀伤剂,其具有由SEQ ID NO:1所表示的氨基酸序列组成的肽。
[2]所述[1]的细胞杀伤剂,其中,所述由SEQ ID NO:1所表示的氨基酸序列组成的肽为下述的肽:
全部由D-氨基酸组成的肽;
在SEQ ID NO:1所表示的氨基酸序列中,第1~14位的氨基酸为止由D-氨基酸组成、第15~19位的氨基酸为止由L-氨基酸组成的肽;
在SEQ ID NO:1所表示的氨基酸序列中,第1~14位的氨基酸为止由L-氨基酸组成、第15~19位的氨基酸为止由D-氨基酸组成的肽;或者
全部由L-氨基酸组成的肽。
[3]所述[1]或者[2]的细胞杀伤剂,其是由细胞异常增殖引起的疾病的治疗剂。
[4]所述[3]的细胞杀伤剂,其中,所述疾病为癌症。
[5]由细胞异常增殖引起的疾病的治疗方法,该治疗方法具有下述的步骤:对于患有由细胞异常增殖引起的疾病的动物,给予有效量的所述[1]~[4]中任一项的细胞杀伤剂。
发明效果
本发明所涉及的细胞杀伤剂可非常有效地针对靶细胞诱导细胞凋亡。因此,该细胞杀伤剂作为癌症等的由细胞异常增殖引起的疾病的治疗剂特别有效。
附图说明
[图1]是显示实施例1中用各肽处理的A431-CNGB3-myc细胞的相对ATP量(%)的测定结果的图。
[图2]是显示实施例1中用各肽处理的OVCAR3-Luc细胞的相对ATP量(%)的测定结果的图。
[图3]是显示实施例1中用各肽处理的OVCAR3-Luc细胞的相对ATP量(%)的测定结果的图。
[图4]是显示实施例2中向腹腔内单次给予了K14D-H5D肽的子宫内膜异位症模型小鼠的腹膜的发光强度(RLU)的测定结果的图。
[图5]是显示实施例3中给予了K14D-H5D肽的OVCAR3-Luc小鼠的经时性地测定光子数的增加率(%)的结果的图。
[图6]是显示实施例3中给予了K14D-H5D肽的OVCAR3-Luc小鼠的经时性地测定肿瘤体积的增加率(%)的结果的图。
[图7]是显示实施例3中给予了K14D-H5D肽的OVCAR3-Luc小鼠的、以肿瘤增加率达到10倍的情况作为安乐死条件的存活率的经时性变化的图。
[图8]是显示实施例3中给予了K14D-H5D肽的OVCAR3-Luc小鼠的、以肿瘤增加率达到20倍的情况作为安乐死条件的存活率的经时性变化的图。
具体实施方式
<细胞杀伤剂>
本发明所涉及的细胞杀伤剂具有由SEQ ID NO:1所表示的氨基酸序列(KLAKLAKKLAKLHLAHL)组成的肽(以下,有时称为“效应肽”)。效应肽是具有细胞凋亡诱导活性的肽与具有内涵体逃逸活性的肽串联地连接而得的肽。本发明所涉及的细胞杀伤剂由于足够小,因此即使不通过特定的摄入***也可通过内吞作用(endocytosis)而摄入到靶细胞内。接着,由于具有内涵体逃逸活性的肽位点的作用,内涵体膜被破坏,其结果,摄入到靶细胞内的内涵体中内含的细胞杀伤剂,被释放到靶细胞的细胞质中。释放到细胞质中的细胞杀伤剂,通过具有细胞凋亡诱导活性的肽位点的作用,阻碍线粒体膜,并诱导该靶细胞的细胞凋亡。
本发明所涉及的细胞杀伤剂在一个分子中包含具有细胞凋亡诱导活性的肽部分和具有内涵体逃逸活性的肽部分。因此,与分别独立地包含具有细胞凋亡诱导活性的肽和具有内涵体逃逸活性的肽的肽组合物相比,可非常有效地对靶细胞诱导细胞凋亡。
需要说明的是,在本发明和本申请说明书中,“靶细胞”是指将要诱导细胞凋亡的目标细胞。
如专利文献1中所公开的那样,由KLAK的4个氨基酸重复而组成的氨基酸序列(以下,有时称为“KLAK序列”)构成的肽(以下,有时称为“KLAK肽”),阻碍线粒体膜而具有诱导细胞凋亡的作用(细胞凋亡诱导活性)。由HLAH的4个氨基酸重复而组成的氨基酸序列(以下,有时称为“HLAH序列”)构成的肽(以下,有时称为“HLAH肽”),具有破坏内涵体膜的作用(内涵体逃逸活性)。将KLAK肽与HLAH肽连接而得的肽,虽然具有内涵体逃逸活性和细胞凋亡诱导活性,但其活性的强度会受到每种肽的氨基酸长度和两种肽的连接顺序的影响。
由SEQ ID NO:1所表示的氨基酸序列组成的效应肽是在由14个氨基酸组成的KLAK序列的下游连接有由5个氨基酸组成的HLAH序列而得的肽。即,在SEQ ID NO:1的氨基酸序列中,第1~14位的氨基酸为止是具有细胞凋亡诱导活性的位点,第15~19位的氨基酸为止是具有内涵体逃逸活性的位点。该效应肽是下述的肽:使KLAK序列的长度、HLAH序列的长度、和KLAK序列与HLAH序列的连接顺序被优化,以使得在通过内吞作用被摄入到靶细胞内的情况下,可得到最高的细胞凋亡诱导活性。由于具有该效应肽,因此本发明所涉及的细胞杀伤剂具有非常高的细胞凋亡诱导活性。
本发明所涉及的细胞杀伤剂所具有的效应肽没有特别限定,只要是由SEQ ID NO:1所表示的氨基酸序列组成的肽即可,可以是由L-氨基酸组成的肽、也可以是由D-氨基酸组成的肽、还可以是由L-氨基酸和D-氨基酸组成的肽。从内涵体内的稳定性高、且可得到更高的细胞凋亡诱导活性的角度考虑,效应肽优选为至少部分包含D-氨基酸的肽,更优选KLAK序列(在SEQ ID NO:1所表示的氨基酸序列中,第1~14位的氨基酸)和HLAH序列(在SEQ IDNO:1所表示的氨基酸序列中,第15~19位的氨基酸)的至少一种序列为由D-氨基酸组成的肽,特别优选为全部由D-氨基酸组成的肽。
关于本发明所涉及的细胞杀伤剂,只要不损及由SEQ ID NO:1所表示的氨基酸序列组成的效应肽的效果,则可在该效应肽上连接其他生物分子。作为其他生物分子可举出肽。但是,优选细胞杀伤剂整体的大小是,即使不通过特定的摄入***也可通过内吞作用而摄入到细胞内的大小。具体而言,该效应肽以外的部分优选1~15个、更优选1~10个左右的肽。在本发明所涉及的细胞杀伤剂包含除由SEQ ID NO:1所表示的氨基酸序列组成的效应肽以外的部分的情况下,除该效应肽以外的部分可以位于该效应肽的N末端侧、也可以位于C末端侧。
本发明所涉及的细胞杀伤剂作为由异常增殖引起的疾病的治疗剂有效,特别优选作为癌症或子宫内膜异位症的治疗剂。对成为本发明所涉及的细胞杀伤剂的治疗目标的癌症的种类没有特别限定,例如可举出:子宫癌、子***、骨盆腔癌、卵巢癌、乳腺癌、腹壁肿瘤、大网膜肿瘤、食道癌、胃癌、小肠癌、结肠癌、直肠癌、盲肠癌、胆囊癌、胰腺癌、肝癌、脾癌、肾癌、舌癌、咽癌、鼻癌、耳下腺癌、甲状腺癌、恶性淋巴瘤、骨肿瘤、皮肤癌、肺癌、纵隔癌、睾丸癌、***癌、膀胱癌、脑肿瘤等。另外,可以是原发癌,也可以是转移性癌。
如后述实施例中所述,本发明所涉及的细胞杀伤剂可针对引起异常增殖的细胞优先地、且非常有效地诱导细胞凋亡。该原因尚未明确,但推测如下。通常,引起异常增殖的细胞,其细胞本身的活性高,通过内吞作用从外部摄入各种物质的活性也高。因此,认为给予到生物体内的本发明所涉及的细胞杀伤剂,与正常细胞相比被优先地摄入到引起异常增殖的细胞内,并诱导细胞凋亡。
在将本发明所涉及的细胞杀伤剂用作药物的情况下,对给予途径(给药途径)没有特别限定,可根据靶细胞和包含该细胞的组织来适当确定。例如,作为本发明所涉及的细胞杀伤剂的给予途径,可举出:口服给予、经静脉给予、腹腔内给予、灌肠给予(直肠给予)等。
由异常增殖引起的疾病的治疗可通过对于患有由细胞异常增殖引起的疾病的动物,给予有效量的本发明所涉及的细胞杀伤剂来进行。该细胞杀伤剂的有效量,对于所给予的动物,只要是与未给予的情况相比可减少动物体内的靶细胞(引起异常增殖的细胞)量的量即可,优选为不会因该细胞杀伤剂而产生严重的副作用的量。该细胞杀伤剂的有效量可考虑靶细胞的种类、给予目标的动物种类、给予方法等通过实验进行确定。例如,在对于患有癌症等的由细胞异常增殖引起的疾病的动物给予的情况下,也可将引起该动物的体内的异常增殖的细胞量与未给予的动物相比,将达到优选90%以下、更优选80%以下、进一步优选75%以下、更进一步优选50%以下的量规定为有效量。
本发明所涉及的细胞杀伤剂可通过常规方法制成散剂、颗粒剂、胶囊剂、片剂、咀嚼剂、缓释剂等口服用固体制剂,溶液剂、糖浆剂等口服用液体制剂,注射剂、灌肠剂、喷雾剂、贴剂、软膏剂等。
本发明所涉及的细胞杀伤剂根据制剂上的需要,可配合赋形剂、粘合剂、润滑剂、崩解剂、流动剂、溶剂、助溶剂、缓冲剂、悬浮剂、乳化剂、等渗剂、稳定剂、防腐剂、抗氧化剂、矫味矫臭剂、着色剂等而进行制剂化。
作为赋形剂,可举出:乳糖、葡萄糖、D-甘露糖醇等糖类,淀粉、结晶纤维素等纤维素类,赤藓糖醇、山梨糖醇、木糖醇等糖醇类,磷酸二钙、碳酸钙、高岭土等。作为粘合剂,可举出:α化淀粉、明胶、***胶、甲基纤维素、羧甲基纤维素、羧甲基纤维素钠、结晶纤维素、D-甘露糖醇、海藻糖、羟丙基纤维素、羟丙基甲基纤维素、聚乙烯吡咯烷酮、聚乙烯醇等。作为润滑剂,可举出:硬脂酸、硬脂酸钙、滑石、蔗糖脂肪酸酯、聚乙二醇等。作为崩解剂,可举出:交联聚维酮(交联聚乙烯吡咯烷酮)、低取代度羟丙基纤维素、淀粉、海藻酸、海藻酸钠等。作为流动剂,可举出:硅酸、硅酸酐、硅酸铝、硅酸钙、硅酸铝镁化合物、氧化铝、氢氧化铝、氧化镁、氢氧化镁等。作为溶剂,可举出:纯化水、生理盐水等。作为助溶剂,可举出:葡聚糖、聚乙烯吡咯烷酮、苯甲酸钠、乙二胺、水杨酰胺、烟酰胺、聚氧乙烯氢化蓖麻油衍生物等。作为缓冲剂,例如可举出:柠檬酸钠水合物、乙酸钠水合物、碳酸氢钠、氨基丁三醇、硼酸、硼砂、磷酸氢钠水合物、磷酸二氢钠等。作为悬浮剂或乳化剂,可举出:十二烷基硫酸钠、***胶、明胶、卵磷脂、单硬脂酸甘油酯、聚乙烯醇、聚乙烯吡咯烷酮、羧甲基纤维素钠等纤维素类、聚山梨醇酯类、聚氧乙烯氢化蓖麻油等。作为等渗剂,可举出:乳糖、葡萄糖、D-甘露醇等糖类、氯化钠、氯化钾、甘油、丙二醇、聚乙二醇、尿素等。作为稳定剂,可举出:聚乙二醇、葡聚糖硫酸钠、亚硫酸钠等。作为防腐剂,可举出:对羟基苯甲酸酯类、氯代丁醇、苄醇、苯乙醇、氯甲酚、脱氢乙酸、山梨酸等。作为抗氧化剂,可举出:亚硫酸盐、抗坏血酸等。作为矫味矫臭剂,可举出:通常在药物和食品领域中使用的甜味剂、香料等。作为着色剂,可举出:通常在药物和食品领域中使用的着色料。
本发明所涉及的细胞杀伤剂可以直接使用、也可以作为包含其他成分的药物用组合物使用。作为该药物用组合物中所含的其他成分,例如可举出:所述的赋形剂、粘合剂、润滑剂、崩解剂、流动剂、溶剂、助溶剂、缓冲剂、悬浮剂、乳化剂、等渗剂、稳定剂、防腐剂、抗氧化剂、矫味矫臭剂、着色剂等。另外,该药物用组合物可含有除本发明所涉及的细胞杀伤剂以外的其他有效成分。
本发明所涉及的细胞杀伤剂优选给予哺乳动物,更优选给予人或小鼠、大鼠、兔、豚鼠、仓鼠、猴、绵羊、马、牛、猪、驴、狗、猫等的家畜或实验动物,进一步优选给予人。
实施例
接下来,示出实施例对本发明进行更详细地说明,但本发明并不限定于以下的实施例。
以下的实验中使用的子宫内膜异位症模型细胞和子宫内膜异位症模型小鼠,通过如下操作进行制作。
<子宫内膜异位症模型细胞(A431-CNGB3-myc细胞)>
作为子宫内膜异位症模型细胞,调制了将编码在C末端融合有myc标签的人CNGB3的基因导入到A431细胞(来源于人上皮样细胞癌的细胞株)并使其强制表达的转化细胞(A431-CNGB3-myc细胞)。
以在DMEM High Glucose培养基(GIBCO公司制)中含有10%灭活FBS(胎牛血清、Corning公司制)和1%青霉素-链霉素(Invitrogen公司制)的培养基作为培养培养基,在37℃、5体积%二氧化碳环境下进行了A431-CNGB3-myc细胞的培养。每2~3天进行传代。
<子宫内膜异位症模型小鼠>
将A431-CNGB3-myc细胞移植到免疫缺陷小鼠(NOD/ShiJic-scid Jcl***、由日本CLEA社提供)的腹腔中,制作了子宫内膜异位症模型小鼠。
具体而言,将冷冻保存的A431-CNGB3-myc细胞进行解冻之后,使用10cm培养皿(Lot No.F3BAXQ103:Thermo Fisher Scientific公司制),进行2次传代。加入培养培养基将该细胞调制成1×106cells/0.5mL/body,将所得的细胞液作为给予液。将该给予液在调制后尽可能快地腹腔内给予7周龄的雌性免疫缺陷小鼠,移植了A431-CNGB3-myc细胞。
需要说明的是,将小鼠在聚碳酸酯笼(W×D×H=270×440×187(mm))内(5~10只/笼)、在19.8~27.1℃、湿度32~75%、12小时照明的环境下进行饲养。使其自由摄取饲料(已灭菌CRF-1(固型)、Oriental酵母工业公司制)和饮料水(已灭菌自来水)。
通过腹膜肿瘤的观察和样本采集来进行模型制作的确认。在自A431-CNGB3-myc细胞移植起1~3周后,每周对2只进行尸检,目视确认在腹膜中是否可观察到肿瘤(1mm左右的粒状),并进行拍照。然后,对腹膜进行样本采集,并切出4个部位(腹部、背部的各左右侧),分别浸入到10%中性缓冲***中进行固定,并冷藏保存。对于***固定后的腹膜,使用抗c-myc抗体进行免疫组织化学染色,研究接种的状态。
A431-CNGB3-myc细胞移植后的小鼠的目视确认的结果,在第1次实验中,在自移植起1周后,虽然目视确认到了肿瘤,但未见到腹膜接种,但是在自移植起2或者3周后,目视确认到了肿瘤和腹膜接种。在第2次实验中,在自移植起1周后,也目视确认到了腹膜接种。另外,移植后小鼠的腹膜组织片的c-myc染色的结果,在自移植起1周后,已经确认到腹膜接种。
<导入荧光素酶基因的卵巢癌荷癌皮下肿瘤小鼠(OVCAR3-Luc小鼠)>
作为要移植到小鼠的肿瘤组织,使用了将导入有荧光素酶基因的卵巢癌细胞株(OVCAR3-Luc细胞、由其他设施转让)进行培养而得的组织。以在RPMImedium 1640(11875-093:Gibco公司制)中含有10%灭活FBS(Corning公司制)的培养基作为培养培养基,在37℃、5体积%二氧化碳环境下进行了OVCAR3-Luc细胞的培养。每2~3天进行传代。
将OVCAR3-Luc细胞株通过胰蛋白酶处理进行剥离,加入培养培养基调制成5×105cells/100μL/body,将所得的细胞液作为给予液。将该给予液在调制后尽可能快地皮下给予8~10周龄的雌性SCID小鼠的背部,由此制作了皮下移植有OVCAR3-Luc细胞的皮下肿瘤模型小鼠。
[实施例1]
对于细胞増殖能力亢进的2种类的癌细胞(A431-CNGB3-myc细胞和OVCAR3-Luc细胞),分别研究了各肽的细胞杀伤活性。所使用的肽的氨基酸序列见表1。在表1所记载的肽中,K14D-H5D肽是全部由D-氨基酸构成的、由SEQ ID NO:1所表示的氨基酸序列组成的肽。K14D肽是全部由D-氨基酸构成的KLAK肽,H5D肽是全部由D-氨基酸构成的HLAH肽。K14D-H5D-Z13肽是在K14D-H5D肽的C末端加成由L-氨基酸构成的Z13肽而得的肽。K7D-H18D肽是全部由D-氨基酸构成的、由SEQ ID NO:5所表示的氨基酸序列组成的肽。
[表1]
<细胞杀伤活性的评价>
使用CellTiter-Glo(注册商标)测定试剂盒(Promega公司制),测定用各肽处理的细胞的ATP量来进行各肽的细胞杀伤活性的评价。
具体而言,首先,将3~4次传代后的A431-CNGB3-myc细胞或者OVCAR3-Luc细胞接种于96孔板(Corning/Costar公司制、Lot No.00515003)中,其中,将细胞的浓度调制成1×104cells/well。自接种起培养2天之后,在各孔中添加各肽,分别使最终浓度达到0(未添加肽)、2.34、4.69、9.38、18.8、37.5、75.0、150.0、或者300.0μM,并培养24小时。需要说明的是,将K14D肽与H5D肽组合,并将两种肽的混合物添加到各孔中。然后,从各孔中去除培养上清,向剩余的细胞中加入CellTiter-Glo缓冲液进行匀浆之后,通过离心回收上清,并将其作为裂解物。在该裂解物中加入2倍量的PBS和与裂解物等量的2×CellTiter Glo试剂进行搅拌,将所得的溶液调制成反应溶液,将该反应溶液在室温下静置了10分钟。使用SynergyH1hybrid multimode microplate reader(多功能微孔板检测仪)(BioTek公司制),测定了室温静置后的各反应溶液的发光强度(Luminescence)(RLU:RELATIVE LIGHT UNITS)。反应溶液的发光强度是ATP量的指标。反应溶液的发光强度越小,ATP量越少,表示所给予的肽的细胞杀伤活性越强。所有试验均以一式三份进行测定,将其平均值作为各反应溶液的发光强度(RLU)。
计算以未添加肽的反应溶液的发光强度(RLU)为100%的情况的各反应溶液的相对发光强度(%)作为相对ATP量(%)。用各肽处理的A431-CNGB3-myc细胞的相对ATP量(%)见图1、用各肽处理的OVCAR3-Luc细胞的相对ATP量(%)见图2和3。图中,“K14D+H5D”显示添加了K14D肽和H5D肽这两种肽的反应溶液的相对ATP量(%)。
如图1和2所示,对于任意一种的癌细胞,在添加了K14D肽和H5D肽这两种肽的情况下,即使各肽的添加量为300μM,也未观察到细胞杀伤活性。相对于此,作为KLAK肽与HLAH肽的融合肽的K14D-H5D肽和K7D-H18D肽显示出:添加量依赖性地降低了相对ATP量,并具有细胞杀伤活性。对于任意一种的癌细胞,K14D-H5D肽的细胞杀伤活性都明显强于K7D-H18D肽。另外,如图3所示,K14D-H5D肽和K14D-H5D-Z13肽对于在细胞表面不具有CNGB3的OVCAR3-Luc细胞的细胞杀伤活性为同等程度。即,确认到:即使是在K14D-H5D肽上连接其他肽而得的肽,也具有细胞杀伤活性,只要是可通过内吞作用摄入的程度的大小即可。
<TUNEL染色>
对于用300μM的K14D-H5D肽处理的A431-CNGB3-myc细胞和未用肽处理的A431-CNGB3-myc细胞,进行了TUNEL(TdT-mediated dUTP nick end labeling)染色。其结果,用K14D-H5D肽处理的A431-CNGB3-myc细胞,几乎所有细胞核被染色成棕色(TUNEL阳性),且与未用肽处理的A431-CNGB3-myc细胞相比,细胞显著减少。相对于此,在未用肽处理的A431-CNGB3-myc细胞中,未见到核被染色成棕色的细胞。由这些结果确认到:K14D-H5D肽诱导细胞凋亡,并显示出细胞杀伤效果。
[实施例2]
对子宫内膜异位症模型小鼠给予实施例1中使用的K14D-H5D肽,研究了对子宫内膜细胞的细胞杀伤活性。
<向腹腔内单次给予肽>
对于自将A431-CNGB3-myc细胞移植到腹腔内起7天后的子宫内膜异位症模型小鼠,在异氟烷麻醉下,将0.5mL加热至37℃的生理盐水溶液装入腹腔内,并在按摩之后立即腹腔内给予将K14D-H5D肽溶解于生理盐水中而得的溶液。给予K14D-H5D肽使得每小鼠体重的给予量达到0mg/10mL/kg(对照)(n=7)、5mg/10mL/kg(n=8)、或者10mg/10mL/kg(n=8)。
<腹膜的采取(样本采集)>
关于给予了K14D-H5D肽的子宫内膜异位症模型小鼠,在自给予肽起24小时后,在异氟烷麻醉下被放血致死,并采取腹膜。将ATP测定用腹膜通过液氮冷冻后,直到测定为止保存在冰柜中。将病理标本制作用腹膜浸入10%中性缓冲***中进行固定之后,冷藏保存。
<体重的测定>
对于各小鼠,在自将A431-CNGB3-myc细胞移植到腹腔内起7天后的给予K14D-H5D肽之前和被放血致死之前,测定了体重。
将K14D-H5D肽的每小鼠体重的给予量为0mg/10mL/kg的小鼠组设为G1组、将为5mg/10mL/kg的小鼠组设为G2组、将为10mg/10mL/kg的小鼠组作为G3组。各组的测定结果(平均值±标准误差)见表2。表中,“体重”栏的“第7天”是自将A431-CNGB3-myc细胞移植到腹腔内起7天后的给予K14D-H5D肽之前的体重,“第8天”是给予K14D-H5D肽之后24小时后的体重。
[表2]
如表2所示,观察到了K14D-H5D肽的给予量依赖性地体重稍微降低的倾向,但该降低量较小。由该结果表明:K14D-H5D肽对正常细胞的影响较小,且该肽可相对安全地给予动物。
<ATP量测定>
ATP量测定使用CellTiter-Glo(注册商标)测定试剂盒(Promega公司制)来进行。测定经冷冻的腹膜的重量,加入冷冻组织的10倍量的CellTiter-Glo缓冲液进行匀浆之后,通过离心回收上清,并将其作为裂解物。在该裂解物中加入2倍量的PBS和与裂解物等量的2×CellTiter Glo试剂进行搅拌,将所得的溶液作为反应溶液,将该反应溶液在室温下静置了10分钟。使用Synergy H1hybrid multimode microplate reader(BioTek公司制)测定了室温静置后的反应溶液的发光强度(RLU)。所有试验均以一式三份(n=3)进行测定,将其平均值作为各反应溶液的发光强度(RLU)的测定值。
各组的ATP量的测定结果(平均值±标准误差)见图4。确认到:所给予的K14D-H5D肽以剂量依赖性地降低了腹膜的发光强度(RLU),且降低了腹膜的ATP量。腹膜的ATP量的降低是指被移植到腹膜的A431-CNGB3-myc细胞的存活细胞数减少。由这些结果确认到:腹腔内的A431-CNGB3-myc细胞被K14D-H5D肽杀伤、即K14D-H5D肽在生物体内环境中发挥细胞杀伤效果。
[实施例3]
对于OVCAR3-Luc小鼠,将实施例1中使用的K14D-H5D肽经静脉给予,研究了对癌细胞的细胞杀伤活性作用。
<经尾静脉给予肽>
对于自将OVCAR3-Luc细胞皮下移植起14天后的OVCAR3-Luc小鼠,1天1次、连续6天经尾静脉给予了50μL将K14D-H5D肽溶解于生理盐水中而得的溶液。给予K14D-H5D肽使得每小鼠体重的给予量达到0μmol/kg/天(对照)、或者1.17μmol/kg/天。
<测定肿瘤组织的大小>
对于各小鼠进行发光成像检测,经时性地测定了背中的肿瘤的光子数和肿瘤组织的大小。
(1)光子数的测定
利用荧光素-荧光素酶发光机制,使用体内发光成像装置进行测定。首先,通过异氟烷吸入麻醉使OVCAR3-Luc小鼠镇静之后,向腹腔内给予了100μL的D-荧光素钾溶解液(将D-荧光素钾(126-05116:和光纯药工业公司制)以最终浓度达到30mg/mL的形式溶解于PBS而得的溶液)。在自给予起15分钟后,通过体内发光成像装置测定该小鼠,并测量了光子数。作为体内发光成像装置,使用了IVIS Imaging System(IVIS200:住商PharmaInternational公司制),其是使用超高灵敏冷却CCD相机的光学定量体内成像设备。
(2)肿瘤体积的测定
通过下述式根据肿瘤的长径和短径求出了各OVCAR3-Luc小鼠的背部的估计肿瘤体积(mm3)。需要说明的是,肿瘤的长径(mm)和短径(mm)使用卡尺进行测定。
[估计肿瘤体积(mm3)]=[长径(mm)]×[短径(mm)]×[短径(mm)]×1/2
图5中示出各小鼠的背中的肿瘤的光子数的增加率(%)的经时性变化,图6中示出各小鼠的背中的肿瘤体积(mm3)的增加率(%)的经时性变化。光子数的增加率和肿瘤体积的增加率均以给予肽溶液的开始日的前一天的值为基准(100%)。图中,“治疗(treatment)”表示经静脉给予了肽溶液的处理期间。另外,在两个图中,误差棒在上方一侧显示标准误差(SEM)。如图5所示,在给予了K14D-H5D肽的小鼠中,光子数几乎没有增加,而且肿瘤体积也几乎没有增加。由这些结果可以说明:通过给予K14D-H5D肽,可杀伤生物体内的肿瘤组织,且该肽可用作抗癌剂。
<存活率的测定>
在将给予肽的前一天的测定值作为比较目标的情况下,将基于光子数测定的肿瘤增加达到10倍或者20倍的情况作为安乐死条件,来计算各小鼠的存活率。进行20天观察,并通过Kaplan-Meier法分析了存活曲线。
将肿瘤增加率达到10倍的情况作为安乐死条件的存活率的经时性变化见图7、将肿瘤增加率达到20倍的情况作为安乐死条件的存活率的经时性变化见图8。如图7和图8所示,通过给予K14D-H5D肽,大幅度改善了OVCAR3-Luc小鼠的存活率,且K14D-H5D肽具有显著的抗肿瘤效果。
序列表
<110> 学校法人藤田学园
<120> 细胞杀伤剂
<130> PC-26985
<160> 5
<210> 1
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 人工序列说明: 效应肽
<400> 1
Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu
1 5 10 15
Ala His Leu
<210> 2
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: K14D 肽
<400> 2
Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys
1 5 10
<210> 3
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工序列说明: H5D 肽
<400> 3
His Leu Ala His Leu
1 5
<210> 4
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 人工序列说明: K14D-H5D-Z13 肽
<400> 4
Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu
1 5 10 15
Ala His Leu Val Arg Arg Ala Asp Asn Arg Pro Gly
20 25
<210> 5
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 人工序列说明: K7D-H18D 肽
<400> 5
Lys Leu Ala Lys Leu Ala Lys His Leu Ala His Leu Ala His His Leu
1 5 10 15
Ala His Leu Ala His His Leu Ala His
20 25
Claims (5)
1.细胞杀伤剂,其具有由SEQ ID NO:1所表示的氨基酸序列组成的肽。
2.权利要求1所述的细胞杀伤剂,其中,所述由SEQ ID NO:1所表示的氨基酸序列组成的肽为下述的肽:
全部由D-氨基酸组成的肽;
在SEQ ID NO:1所表示的氨基酸序列中,第1~14位的氨基酸为止由D-氨基酸组成、第15~19位的氨基酸为止由L-氨基酸组成的肽;
在SEQ ID NO:1所表示的氨基酸序列中,第1~14位的氨基酸为止由L-氨基酸组成、第15~19位的氨基酸为止由D-氨基酸组成的肽;或者
全部由L-氨基酸组成的肽。
3.权利要求1或2所述的细胞杀伤剂,其为由细胞异常增殖引起的疾病的治疗剂。
4.权利要求3所述的细胞杀伤剂,其中,所述疾病为癌症。
5.由细胞异常增殖引起的疾病的治疗方法,该治疗方法具有下述的步骤:对于患有由细胞异常增殖引起的疾病的动物,给予有效量的权利要求1~4中任一项所述的细胞杀伤剂。
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CN103435683A (zh) * | 2006-06-16 | 2013-12-11 | 肿瘤疗法·科学股份有限公司 | 来自sparc的癌排斥抗原肽以及含有该肽的药物 |
US20120251453A1 (en) * | 2009-12-23 | 2012-10-04 | Michiko Fukuda | Methods and Compositions Related to Annexin 1-Binding Compounds |
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