CN116789737A - 一种靶向cd38的多肽及其应用 - Google Patents
一种靶向cd38的多肽及其应用 Download PDFInfo
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Abstract
本发明涉及生物医学技术领域,尤其涉及一种靶向CD38的多肽及其应用。本发明提供了靶向CD38的多肽,该多肽特异性高、敏感性好,分子量小,生物安全性高,免疫原性低,肿瘤渗透性高。该多肽可以采用化学合成的方法合成,操作简单,生产成本低。本发明的小分子多肽更易于药物设计和修饰,可进一步优化成为多功能的靶向材料,有很强的实用性和应用前景。将本发明的多肽与显像剂结合制备成分子探针,能够克服单抗分子探针的分子量大、易失活、组织渗透慢、血液清除慢等缺陷。本发明的多肽及其衍生产品在肿瘤治疗、诊断、成像中具有极好的应用前景。
Description
技术领域
本发明涉及生物医学技术领域,尤其涉及一种靶向CD38的多肽及其应用。
背景技术
多发性骨髓瘤(multiple myeloma,MM)是一种年龄相关的骨髓浆细胞异常增殖且伴有单克隆免疫球蛋白或轻链(M蛋白)过度生成的恶性疾病。MM多发于老年人,起病隐匿,多数患者确诊时已是中晚期。随着人口的老龄化,MM的发病率呈上升趋势,目前已超过急性白血病。其临床表现是骨质破坏,缺乏特异性,诊断主要依赖于骨髓活检中克隆性骨髓浆细胞增多,属于有创性检查,且骨穿位置选择不当会导致假阴性的结果。CD38是一种多功能的跨膜糖蛋白,具有酶和受体的作用。CD38在MM的恶性浆细胞表面呈显著高表达,使其成为MM特征性的肿瘤生物靶标。此外,CD38在肿瘤微环境的相互作用、免疫抑制活性的调节以及淋巴瘤和其他恶性肿瘤的免疫激活中起着重要作用。
达雷木单抗(daratumumab)这一靶向CD38表位的人源化免疫球蛋白G1-kappa(IgG1-κ)抗体,可以与细胞表面CD38抗原特异性结合并诱导肿瘤细胞死亡,在复发和难治性MM患者中显示出良好的疗效和安全性,但一些初期疗效良好的患者可能会产生反应性减弱甚至耐药。而且,基于完整抗体的成像/诊断试剂的一个主要限制是血循环半衰期过长。除此之外,抗体的制备过程复杂,成本较高,导致治疗费用昂贵。且抗体作为生物大分子,实体瘤的穿透性差,免疫原性强,临床上有许多不可避免的副作用。
发明内容
本发明通过对CD38和daratumumab复合物晶体结构进行分析,提取结合的热点氨基酸位点,进行单点突变,计算机模拟辅助设计出肽库,结合Docking打分和结合能大小,初步筛出候选肽。随后,利用表面等离子共振技术(SPRi)筛选出一系列CD38高亲和性多肽。
经筛选后最终获得了本发明的靶向CD38的多肽。本发明提供的靶向CD38的多肽特异性高、敏感性好,分子量小,生物安全性高,免疫原性低,肿瘤渗透性高,并选择性结合CD38高表达的肿瘤细胞。基于此,特提出以下发明。
第一方面,本发明提供了一种靶向CD38的多肽,其氨基酸序列为SEQ ID No.1所示。
第二方面,本发明提供了上述多肽的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物;
所述衍生物为所述多肽形成的二价体或多价体。
本发明中,所述氨基酸的残基可以是L-型,D-型,或L-型与D-型的混合。
本发明中,所述异构体包括氨基酸序列前后颠倒的序列变化体或环肽结构。
本发明中,所述二价体或多价体能够靶向CD38。
优选地,所述的二价体或多价体通过连接分子经共价连接或非共价连接形成,或者通过与多聚体混合经非共价连接形成。
优选地,所述共价连接的连接分子为异硫氰酸荧光素、6-叔丁氧羰肼基烟酸、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺、N-羟基琥珀酰亚胺、1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸中的至少一种。
优选地,所述非共价连接的连接分子包括但不限于亲脂性近红外染料。
优选地,所述多聚体为聚乙二醇(PEG)、聚乙烯醇(PVA)、环糊精、聚酰胺-胺型树枝状高分子(PAMAM)、聚乳酸(PLA)、聚乳酸-乙醇胺(PLGA)中的至少一种。
第三方面,本发明提供了编码所述多肽的核酸。
第四方面,本发明提供了一种生物材料,其包括所述多肽、所述多肽的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或所述的核酸;优选地,所述生物材料为载体、表达盒、转座子、宿主细胞或转基因细胞系。
本发明中,所述载体包括但不限于克隆载体、表达载体、质粒载体,所有包含至少一个拷贝的所述编码本发明所述靶向CD38多肽的核酸的载体均在本发明的保护范围内。所述宿主细胞或转基因细胞系可以为来源于微生物、植物或动物的细胞或细胞系,所有含有至少一个拷贝的所述编码本发明所述靶向CD38多肽的核酸或包含携带至少一个拷贝的所述核酸的载体的宿主细胞或转基因细胞系均在本发明的保护范围内。
优选地,本发明所述的靶向CD38的多肽采用Fmoc固相多肽合成法制备得到。
第五方面,本发明提供了一种药物,其包括所述多肽、所述多肽的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或所述的核酸、或所述的生物材料以及药学上可接受的辅料。
在一些实施方案中,所述药物还包括杀伤肿瘤细胞的制剂。
优选地,所述杀伤肿瘤细胞的制剂为杀伤肿瘤细胞的化学药物、生物药物、纳米药物、放射性药物、光热治疗或光动力治疗药物中的至少一种;
或为烷化剂、抗代谢药物、抗肿瘤天然药物、抗肿瘤抗生素、激素、金属络合物或肿瘤放射靶向标记物中的至少一种。
在一些实施方案中,所述药物还包括药物载体。
所述载体包括但不限于用于制备靶向药物的载体。
优选地,所述载体为纳米材料、脂质体或油性化合物中的至少一种。
所述药学上可接受的辅料包括佐剂。
本发明的上述药物可用于多种肿瘤的靶向治疗和联合治疗。
第六方面,本发明提供了一种偶联物,其包括载体和选自以下组分中的至少一种:所述的多肽、或所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或所述的核酸、或所述的生物材料。
在所述偶联物中,所述载体与上述组分以共价或非共价的方式连接或作用得到。
优选地,所述载体为荧光素、抗体、多聚物、高分子材料、纳米材料、脂质体、油性化合物、无机材料中的任意一种或多种。
进一步优选地,所述高分子材料为聚酯、聚酸酐、聚酰胺磷脂聚合物胶束、聚乳酸-羟基乙酸共聚物、聚乙二醇、壳聚糖中的至少一种。
进一步优选地,所述无机材料为纳米金、碳材料、钙材料、磁性材料、介孔硅材料、量子点中的至少一种。
第七方面,本发明提供了一种显像制剂,其包括成像剂和选自以下组分中的至少一种:所述的多肽、或所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或所述的核酸、或所述的生物材料、或所述的药物、或所述的偶联物;
所述成像剂为放射性核素、放射性核素标记物、荧光分子、磁共振造影剂或分子影像制剂中的至少一种。
在所述显像制剂中,所述成像剂和所述组分以相偶联或混合物的形式存在。
本发明的显像制剂可以实时监控免疫治疗的疗效,可以作为CD38免疫治疗的预测和伴随诊断试剂。
第八方面,本发明提供了一种试剂或试剂盒,其包括所述的多肽、或所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或所述的核酸、或所述的生物材料、或所述的药物、或所述的偶联物、或所述的显像制剂。
第九方面,本发明提供了所述的多肽、或所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或所述的核酸、或所述的生物材料、或所述的药物、或所述的偶联物、或所述的显像制剂、或所述的试剂或试剂盒在以下至少一方面的应用:
(1)检测细胞CD38表达水平;
(2)制备检测细胞CD38表达水平的试剂;
(3)制备药品;所述药品用于诊断、预防或治疗CD38表达异常的疾病;
(4)药物载体。
优选地,所述CD38表达异常的疾病为肿瘤、类风湿性关节炎、动脉粥样硬化、冠心病中的至少一种。
更优选地,所述肿瘤为急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)、急性髓性白血病(acute myeloid leukemia,AML)、慢性淋巴细胞白血病(chroniclymphocytic leukemia,CLL)、非霍奇金淋巴瘤(non-Hodgkin lymphoma,NHL)中的至少一种。
与现有技术相比,本发明的有益效果在于:
本发明的多肽特异性高、敏感性好,分子量小,生物安全性高,免疫原性低,肿瘤渗透性高。该多肽可以采用化学合成的方法合成,操作简单,生产成本低。本发明的小分子多肽更易于药物设计和修饰,可进一步优化成为多功能的靶向材料,有很强的实用性和应用前景。将本发明的多肽与显像剂结合制备成分子探针,能够克服单抗分子探针的分子量大、易失活、组织渗透慢、血液清除慢等缺陷。本发明的多肽及其衍生产品在肿瘤治疗、诊断、成像中具有极好的应用前景。
附图说明
图1是本发明CP1多肽固相合成后的质谱鉴定图。
图2是本发明提供的CP1多肽与人CD38蛋白的亲和力的表面等离子共振结果图。
图3是本发明提供的FITC标记多肽与CD38阳性细胞系Ramos以及阴性细胞系U266的特异亲和性检测结果图。
图4是多肽探针的小动物体内荧光成像。
图5是68Ga标记的CD38靶向多肽的标记率和放化纯HPLC测定结果图。
图6是小动物PET成像显示68Ga标记的CD38靶向多肽在CD38阳性肿瘤Ramos以及阴性肿瘤U266的活体特异检测结果图。
图7是68Ga标记的CD38靶向多肽在CD38阳性肿瘤Ramos以及阴性肿瘤U266的SUV对比结果图。
图8是CD38阳性肿瘤Ramos以及阴性肿瘤U266小鼠中各脏器的放射性分布摄取的差异统计图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例中未注明具体技术或条件者,均为常规方法或者按照本领域的文献所描述的技术或条件进行,或者按照产品说明书进行。所用试剂和仪器等未注明生产厂商者,均为可通过正规渠道商购买得到的常规产品。
实施例1CD38靶向多肽的制备方法
通过微芯片基于的组合化学肽库筛选获得后采用标准Fmoc方案,利用固相合成方法合成多肽。将目标多肽的C-端羧基以共价键形式与高分子树脂相连,然后以这个氨基酸的氨基作为起点,与另一氨基酸的羧基作用形成肽键。固相合成顺序从C端向N端,不断重复上一步骤,将单个氨基酸逐个偶联到固相树脂上,直到得到目标多肽产物。反应完成后,去除保护基,用裂解液将肽链与树脂分离,即得到目标产物。MALDI-TOF鉴定和HPLC纯化用于后续试验。经化学合成制得本发明的优选多肽CP1(SEQ ID No.1),质谱鉴定结果如图1所示,表明该靶向多肽的正确性。
实施例2CP1多肽与人CD38蛋白的亲和作用
本实施例通过表面等离子共振(SPRi)方法检测CP1多肽与人CD38蛋白的亲和作用。具体地:
将CP1多肽溶液点到SPRi芯片上,4℃湿润条件下孵育过夜,10×PBS清洗10min,1×PBS清洗10min,去离子水清洗2次,每次10min,5%脱脂牛奶封闭过夜,重复上述清洗步骤,随后氮气吹干,等待上机(PlexeraHT表面等离子共振成像系统)。
流动相依次通过1×PBS、2×PBS、16.5nM、33nM、66nM、132nM和264nM的CD38蛋白样品,记录分析SPRi信号。
由图2可以看出,CP1的SPRi信号随着蛋白浓度的增加逐渐增强,且KD值达到10-8M,说明本发明的多肽对CD38有很强的亲和力,可以满足后续的体内应用。
实施例3FITC标记多肽的制备
多肽异硫氰酸荧光素(FITC)偶联物采用固相合成方法获得,在固相合成的多肽树脂上继续偶联ε-氨基己酸。在吡啶/N,N二甲基甲酰胺/二氯甲烷比例为1:5:7的溶液中,将FITC与肽珠混合反应过夜,注意避光。经裂解液裂解后得到多肽FITC偶联物,采用MALDI-TOF鉴定和HPLC纯化用于后续实验。
实施例4细胞水平验证CD38靶向多肽的亲和力
实验选择的阳性细胞为Ramos,阴性细胞为U266,用含10%FBS的RPMI1640培养基培养。
将目标多肽分别与阳性细胞和阴性细胞相互作用,用共聚焦显微技术探究其结合情况。具体实验步骤为:将两种细胞分别种植到共聚焦小皿上,37℃,5% CO2细胞培养箱中培养过夜,贴壁,弃去培养液,用Hoechst 33342试剂进行细胞染核,室温孵育10min,PBS清洗2次。随后加入0.1mg/mL的多肽FITC偶联物,4℃避光孵育20min,PBS清洗3次。用激光扫描共聚焦显微镜(ZEISS LSM 710)检测细胞中的荧光分布情况。
结果如图3所示,Ramos细胞膜有明显的FITC的绿色荧光信号,而U266细胞膜几乎没有荧光。结果表明目标多肽均能有效结合在CD38高表达的肿瘤细胞的细胞膜上,而不能结合低表达的肿瘤细胞,可见,CP1多肽对CD38的识别具有特异性,与分子水平的SPRi数据相符合。
实施例5多肽探针的体内高灵敏荧光成像
将Ramos细胞用含10%胎牛血清的RPMI1640培养基培养,按1×106通过皮下注射入Balb/c鼠右后肢,待肿瘤长至50mm3。称取1mg ICG-CP1多肽溶于1mL的1×PBS中,尾静脉注射150μL的ICG-CP1多肽探针半小时后,使用IVIS Spectrum小动物活体光学三维成像系统进行信号采集,24h解剖后取主要器官进行体外荧光分布成像。
结果如图4所示,将上述制备得到的多肽显像制剂经尾静脉注射入CD38阳性的荷瘤小鼠体内,在0.5小时之内,肿瘤部位荧光信号逐渐增强,2h荧光信号最强,随后逐渐减弱持续到8h,与对照单独染料比,解剖后肿瘤也有很强的荧光信号,同时肝肾也有较强的富集。上述结果证明了本发明的多肽小分子探针具有快速靶向CD38靶向性,同时具有快速清除代谢能力,可以实现微小肿瘤的高灵敏度活体成像。
实施例6多肽探针在荷瘤小鼠模型的ImmunoPET显像
实施例中所有动物实验均按照北京大学第一医院动物管理和使用委员会批准的方案进行。
选择具有T细胞和B细胞双重缺陷的CB17-SCID免疫缺陷小鼠(4-6周龄,雄性)用于淋巴瘤皮下肿瘤模型的构建,将小鼠随机分为两组,即实验组和对照组,每组5只。CB17-SCID免疫缺陷小鼠为北京维通利华实验动物技术有限公司产品。于每只小鼠右侧腋窝皮下注射含1×107个Ramos和U266淋巴瘤细胞的200μL Matrigel混悬液(Invitrogen,USA)。隔天监测小鼠的健康状况和肿瘤体积。当肿瘤直径达1cm时,可用于活体显像和生物分布实验。
使用0.6M高纯盐酸淋洗68Ge-68Ga锗镓发生器得到GaCl3溶液,取1mL Ga-68溶液,加入100微升氢氧化钠(3M),130微升醋酸钠(3M),盖上盖子,混匀,用0-6精密pH试纸测pH为4-4.5,将要标记的分子加入已经调好的溶液。90℃加热十分钟后洗脱。冷却反应液后,将其加入活化后的Sep-Pak LightC18小柱(5mL乙醇,5mL去离子水,按照乙醇-水-空气活化)。以3.0mL纯水淋洗杂质并弃去。加上0.22μm无菌微孔滤膜,以0.5mL乙醇溶液收集产品到无菌真空瓶中,向体系中加入5.0mL生理盐水,待用。
68Ga-DOTA-CD38多肽标记产物进一步通过高效液相色谱法测定,色谱条件:色谱柱为C18色谱柱(4.6×150mm,5μm,XBridge,Waters),流动相A相为去离子水(0.1%三氟乙酸),B相为乙腈(0.1%三氟乙酸),流速为1.0毫升每分钟。具体分析方法为:0-2分钟,10%B;2-10分种,10%-60%B;10-12分钟,60% B;12-15分钟,60%-10%B。收集放射性谱图。
结果如图5所示,制剂放射性色谱峰保留时间差别不大于0.5min,68Ga-DOTA-CD38多肽标记产物放射化学纯度接近99.9%。
待荷瘤小鼠肿瘤直径长至约1cm时,分别尾静脉注射5-10MBq的68Ga-DOTA-CD38多肽探针。注射后30min、40min、50min、60min、70min、80min使用Inveon micro-PET/CT扫描仪(Siemens,Germany)进行PET显像。通过使用Inveon软件绘制感兴趣区域(region ofinterest,ROI)并进行定量分析,获得活体中不同时间点肿瘤、心血池(血液)、肝脏、肾脏内的放射性浓聚情况。放射性探针的含量用每克组织的放射性计数占总注入的放射性计数的百分比(%ID/g)表示,代表放射性摄取量。
结果如图6所示,最大密度投影(maximum intensity projection,MIP)PET显像结果显示,在CD38表达阳性的Ramos肿瘤模型中,在68Ga-DOTA-CD38多肽注射后30min,肿瘤即可见明显的放射性浓聚,且从30分钟到80分钟均有较高的摄取。而在CD38表达阴性的U266肿瘤模型中,注射68Ga-DOTA-CD38多肽探针后各时间点,肿瘤均未见明显放射性浓聚。
通过使用相应配套软件绘制感兴趣区域(region of interest,ROI)并进行定量分析,获得活体中不同时间点肿瘤的放射性浓聚情况。
结果如图7所示,Ramos肿瘤在30分钟到80分钟内均保持较高放射性摄取,且均高于阴性对照组。
在注射68Ga-DOTA-CD38多肽探针后后,解剖肿瘤、血液、心、肝、脾、肺、肾、胃、胰腺、小肠、膀胱、肌肉、小腿长骨、脑、尾巴,样品称重,并使用自动γ计数器(PerkinElmer)对样品放射性进行计数,计算不同器官/组织中放射性示踪剂的摄取量并计算生物分布,并以%ID/g(平均值±SD)表示。
生物分布结果如图8所示,Ramos肿瘤摄取(0.75±0.03%ID g-1)高于U266肿瘤(0.26±0.08%ID g-1,p<0.01)。此外,Ramos荷瘤小鼠中肺的放射性示踪剂的摄取量为8.92%ID g-1,肝的摄取量为4.03±0.09%ID g-1,肾的摄取量为18.38±0.82%ID g-1,均高于U266荷瘤小鼠各个器官的摄取(分别为3.67±0.51%ID g-1,1.47±0.25%ID g-1,12.41±3.39%ID g-1),与PET显像结果一致。
上述结果证明了本发明的多肽小分子探针具有快速靶向CD38靶向性,同时具有较好的肿瘤穿透能力,可以实现微小肿瘤的高灵敏度活体成像。
综上所述,本发明的多肽具有靶向表达CD38阳性肿瘤细胞的特性,因而在实际应用中,可以将本发明的多肽作为归巢肽,与抗癌药物或者显像剂结合,用于肿瘤的靶向治疗和成像。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种靶向CD38的多肽,其特征在于,其氨基酸序列为SEQ IDNo.1所示。
2.权利要求1所述多肽的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物;
所述衍生物为所述多肽形成的二价体或多价体。
3.编码权利要求1所述多肽的核酸。
4.一种生物材料,其特征在于,其包括权利要求1所述的多肽、或权利要求2所述的多肽的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或权利要求3所述的核酸;优选地,所述生物材料为载体、表达盒、转座子、宿主细胞或转基因细胞系。
5.一种药物,其特征在于,其包括权利要求1所述的多肽、或权利要求2所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或权利要求3所述的核酸、或权利要求4所述的生物材料以及药学上可接受的辅料。
6.一种偶联物,其特征在于,其包括载体和选自以下组分中的至少一种:权利要求1所述的多肽、或权利要求2所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或权利要求3所述的核酸、或权利要求4所述的生物材料。
7.根据权利要求6所述的偶联物,其特征在于,所述载体为荧光素、抗体、多聚物、高分子材料、纳米材料、脂质体、油性化合物、无机材料中的任意一种或多种。
8.一种显像制剂,其特征在于,其包括成像剂和选自以下组分中的至少一种:权利要求1所述的多肽、或权利要求2所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或权利要求3所述的核酸、或权利要求4所述的生物材料、或权利要求5所述的药物、或权利要求6所述的偶联物;
所述成像剂为放射性核素、放射性核素标记物、荧光分子、磁共振造影剂或分子影像制剂中的至少一种。
9.一种试剂或试剂盒,其特征在于,其包括权利要求1所述的多肽、或权利要求2所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或权利要求3所述的核酸、或权利要求4所述的生物材料、或权利要求5所述的药物、或权利要求6所述的偶联物、或权利要求8所述的显像制剂。
10.权利要求1所述的多肽、或权利要求2所述的异构体、衍生物、药学上可接受的盐、水合物或溶剂化物、或权利要求3所述的核酸、或权利要求4所述的生物材料、或权利要求5所述的药物、或权利要求6所述的偶联物、或权利要求8所述的显像制剂、或权利要求9所述的试剂或试剂盒在以下至少一方面的应用:
(1)检测细胞CD38表达水平;
(2)制备检测细胞CD38表达水平的试剂;
(3)制备药品;所述药品用于诊断、预防或治疗CD38表达异常的疾病;
(4)药物载体。
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