CN117003852A - 白细胞介素-2的拓扑改造及其作为自身免疫病药物的应用 - Google Patents
白细胞介素-2的拓扑改造及其作为自身免疫病药物的应用 Download PDFInfo
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Abstract
本发明公开了白细胞介素‑2的拓扑改造及其作为自身免疫病药物的应用。本发明对白细胞介素‑2进行拓扑改造,获得了靶向性激活Treg的环状或索烃拓扑结构的白细胞介素‑2。环状或索烃结构的白细胞介素‑2不改变其与受体的亲和性,同时可以延长药物的半衰期,增强稳定性,减少长期用药的剂量和用药次数,发挥抑制免疫的效果。本发明的白细胞介素‑2拓扑改造蛋白作为长效的免疫抑制剂,有利于治疗多种自身免疫病。
Description
技术领域
本发明属于生物制药领域,具体涉及拓扑改造的白细胞介素-2,及其在自身免疫病治疗中的应用。
背景技术
白细胞介素-2(IL-2)是一种多功能T细胞生长因子,对促进传统辅助性T细胞(Tcon)、效应T淋巴细胞(Teff)及CD8+T细胞增殖至关重要。高剂量的IL-2用于治疗肿瘤,增强癌症患者的免疫原性;低剂量IL-2可以选择性地刺激调节T细胞(Treg)的增殖,抑制Tcon和Teff细胞的分化,有效改善系统性红斑狼疮(SLE)患者的免疫稳态。经低剂量IL-2治疗后,患者的皮疹、发热、关节炎、肾炎等临床症状及抗dsDNA的滴度和补体水平等均显著改善,同时,患者体内Treg水平升高,且抑制功能增强,致病性Tfh和Th17细胞水平降低,免疫失衡改善,反映病情程度的SLEDAI评分显著下降。但是,自身免疫病患者多需长期用药,目前使用的IL-2剂型并非针对自身免疫病的低剂量IL-2疗法设计,半衰期较短。基于以上原因,需要具有改进的药代动力学和药效长久的IL-2生物制剂。
发明内容
为了解决上述问题,本发明的目的是提供靶向调节T细胞(Treg)的长效白细胞介素-2及其在治疗自身免疫病中的应用。
为实现上述技术目的,本发明对白细胞介素-2(IL-2)进行了拓扑改造,以提供可使白细胞介素-2靶向性激活Treg的环状及索烃拓扑结构。本发明经拓扑改造得到的环状白细胞介素-2和索烃白细胞介素-2的结构参见图1,分别如下(1)和(2)所述:
(1)环状改构蛋白c-IL-2:白细胞介素-2的N端和C端相连,形成具有环状结构的白细胞介素-2;
(2)索烃改构蛋白cat-(X-IL-2):IL-2与肿瘤抑制因子p53dim(X)等能够形成二聚体的蛋白质缠结基元融合表达,通过肿瘤抑制因子p53dim(X)等蛋白质缠结基元形成分子间的缠结二聚体并形成闭环结构,得到具有索烃结构的X-IL-2二聚体。
环状白细胞介素-2和索烃白细胞介素-2的构建可参考中国发明专利ZL.201510593863.9、ZL.2019113736.3.5、ZL.202010436910.X等记载的方法。将蛋白质偶联反应对连接在白细胞介素-2的N端和C端,使表达出的蛋白质前驱体序列在胞内发生偶联反应实现环化,得到环状结构的白细胞介素-2。蛋白质偶联反应对可以由分离型内含肽的C端部分(IntC)和N端部分(IntN)组成,也可以由谍标签和谍捕手组成。进一步的,将IL-2与能够形成二聚体的蛋白质缠结基元融合表达,并在融合表达片段的N端和C端连接蛋白质偶联反应对,表达出的蛋白质前驱体序列在胞内形成二聚体并关环,得到索烃结构的白细胞介素-2二聚体。
在本发明的实施例中,改造成环状结构的人白细胞介素-2的氨基酸序列如序列表中SEQ ID No:1所示;改造成索烃结构的人白细胞介素-2由两个相同的环构成,每个环的氨基酸序列如序列表中SEQ ID No:2所示。
本发明还提供了上述IL-2拓扑改造蛋白在制备治疗免疫相关疾病的药物中的应用,可以针对系统性红斑狼疮、类风湿关节炎、干燥综合征等自身免疫性疾病进行安全、有效、单次长效的治疗,也可应用于其他相同发病机制的疾病,包括脏器移植排斥反应、移植物抗宿主疾病、皮肌炎、多发性硬化等,以及肿瘤、病毒感染、免疫缺陷病的药物治疗。
含有上述IL-2拓扑改造蛋白的药物组合物也在本发明的保护范围内。所述药物组合物可采用注射剂、片剂、散剂、胶囊剂、膜剂、栓剂等剂型,还可以加入一种或多种药学上可接受的载体或辅料。所述载体或辅料包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂、吸收促进剂、吸附载体等。
基于本发明得到的IL-2拓扑改造蛋白具有化学性质和生物活性均一的特点,与现有研究药物(天然IL-2或重组IL-2)相比,具有以下的技术优势:
本发明通过对IL-2进行拓扑改造,在保留IL-2靶向性的同时,提高了IL-2的半衰期,增强稳定性及其与受体的亲和性,减少IL-2长期用药的剂量和给药次数,提高了IL-2治疗的安全性。
附图说明
图1所示为环状白细胞介素-2(A)和索烃白细胞介素-2(B)的基因卡带及其模拟蛋白质结构。将环状和索烃白细胞介素-2的重组质粒转入大肠杆菌Origami(DE3)中,通过诱导表达,分离型内含肽在胞内反应关环,得到具有特定拓扑结构的白介素-2。图中所示为不同拓扑结构的表达产物及其与受体IL-2Rα、IL-2Rβ、IL-2Rγ之间的关系。
图2所示为环状白细胞介素-2(c-IL-2)和索烃白细胞介素-2(cat-(X-IL-2))的性质表征,其中:(A)为SDS-PAGE表征,由于闭环的蛋白质相较于线性蛋白质更为紧凑,迁移速率更快,故表现为较小的表观分子量;(B)为尺寸排阻色谱表征,索烃白细胞介素-2的水合半径较环状分子略大,故保留体积较小,符合预期;(C)为LC-MS表征,蛋白质的实际分子量得到验证;(D)为圆二色谱表征,证明环状和索烃异构体具有和野生型类似的二级结构。
图3为野生型白细胞介素-2(wt-IL-2)、环状白细胞介素-2(c-IL-2)和索烃白细胞介素-2(cat-(X-IL-2))的半衰期情况,c-IL-2和cat-(X-IL-2)的半衰期明显长于wt-IL-2。
图4为c-IL-2和cat-(X-IL-2)与IL-2Rα和IL-2Rβ的亲和力表征,c-IL-2和cat-(X-IL-2)与IL-2Rα结合的KD在10-9M级别,与IL-2Rβ结合的KD在10-7M级别。
图5为干燥综合征(SS)、系统性红斑狼疮(SLE)、类风湿关节炎(RA)患者外周血对IL-2拓扑改构药物的体外细胞分化检测。系统性红斑狼疮、类风湿关节炎和干燥综合征患者的Treg、Tfh、Th1、Th2、Th17细胞在不同拓扑药物处理后的升高和抑制情况表明,在上述患者中c-IL-2及cat-(X-IL-2)可较高水平地激活Treg细胞,并抑制Tfh及Th1细胞。
图6为IL-2拓扑改构药在干燥综合征自发鼠动物模型体内的免疫反应。检测表明,c-IL-2及cat-(X-IL-2)对免疫调节的Treg有明显的上调作用。
图7为IL-2拓扑改构药在干燥综合征动物模型中的有效性评价,图示为治疗后小鼠的唾液流率,检测表明注射c-IL-2后小鼠唾液流率有明显升高且与WT组(IL-2)持平,cat-(X-IL-2)组唾液流率有上升趋势但无明显差异。
图8为IL-2拓扑改构药在系统性红斑狼疮动物模型体内的免疫反应。c-IL-2及cat-(X-IL-2)对免疫调节的Treg有明显的上调作用,其诱导小鼠产生淋巴结Treg细胞增殖明显高于WT组(IL-2),抑制生发中心B细胞增殖作用与WT组持平。
图9为自身免疫性疾病小鼠模型HE染色图,给予系统性红斑狼疮鼠两种拓扑改构药、IL-2及PBS刺激。检测发现在c-IL-2和cat-(X-IL-2)炎症聚集明显低于空白组(PBS),且与WT组(IL-2)差别不大。
图10为治疗后抗核抗体(ANA)含量。c-IL-2及cat-(X-IL-2)处理系统性红斑狼疮模型鼠后检测表明,注射c-IL-2及cat-(X-IL-2)后小鼠的ANA含量明显低于空白组(PBS)。
图11为IL-2拓扑改构药在系统性红斑狼疮动物模型中的有效性评价,图示为治疗后小鼠的尿蛋白量,给予药物刺激后,c-IL-2及cat-(X-IL-2)组的小鼠尿蛋白明显降低,且比WT组(IL-2)下降明显。
图12为拓扑IL-2对MRL/lpr小鼠的毒性评价。给予药物刺激后,小鼠均无肺水肿迹象,且各项水平均在正常范围内,说明拓扑IL-2没有明显毒性。
图13为IL-2拓扑改构药在胶原诱导的关节炎动物模型中的有效性评价。给予小鼠两种拓扑改构药、IL-2及PBS刺激。对小鼠关节炎严重情况进行评分:0=无红肿;1=小趾关节肿胀;2=趾关节和组织部肿胀;3=踝关节以下的足爪肿胀;4=包括踝关节在内的全部足爪肿胀。图示c-IL-2及cat-(X-IL-2)组的小鼠关节炎严重程度明显下降,且比WT组下降明显。
图14为IL-2拓扑改构药在类风湿关节炎动物模型体内的免疫反应。检测表明,注射c-IL-2及cat-(X-IL-2)的实验组小鼠淋巴结Treg细胞显著高于PBS组,且略高于WT组(IL-2);脾脏Treg细胞也显著高于PBS组;注射c-IL-2及cat-(X-IL-2)的实验组小鼠淋巴结Th1、Th2、Th17细胞显著低于PBS组,c-IL-2及cat-(X-IL-2)的实验组小鼠淋巴结Th2细胞显著低于WT组小鼠。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1、环状及索烃白细胞介素-2的制备
根据IL-2的晶体结构,本发明在IL-2的两端分别融合分离型内含肽的C端(IntC)和N端(IntN)部分,得到IntC-IL-2-IntN重组质粒,如图1中(A)所示;在上述质粒中插入p53dim(X),得到IntC-X-IL-2-IntN重组质粒,如图1中(B)所示。两者的表达载体皆为pET15b。将上述带有氨苄青霉素抗性的表达质粒转化到大肠杆菌Origami(DE3)中,经氨苄抗性平板筛选出阳性菌株。获得的阳性表达菌株在含100μg/mL氨苄青霉素的2×YT培养基中37℃培养12小时,接种扩大培养至菌液OD到0.6-0.8时,加入IPTG至终浓度0.3mM,16℃诱导表达12小时后收集菌体。菌体用裂解缓冲液重悬,经细胞破碎仪处理后,高速离心除去细胞碎片。使用Ni-NTA亲和层析法进行纯化,先用漂洗缓冲液充分洗涤,再用洗脱缓冲液洗脱,得到初步纯化的c-IL-2及cat-(X-IL-2)样品。产物经尺寸排阻色谱纯化和表征,纯化样品经LC-MS和圆二色谱表征其分子量和二级结构,如图2所示。
c-IL-2(148aa)的氨基酸序列(SEQ ID No:1)如下:
CFNGGHHHHHHELAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST LTGT(下划线部分为野生型白细胞介素-2的氨基酸序列)
cat-(X-IL2)(388aa)由两个相同的环构成,每个环的氨基酸序列(SEQ ID No:2)如下:
(斜体部分为p53dim序列,下划线部分为野生型白细胞介素-2序列)
实施例2、拓扑IL-2的半衰期及其与受体的亲和力表征
将六周龄雌性KM小鼠随机分为三组(n=5),每组以60μg IL-2/kg体重的剂量进行注射,将WT组IL-2(欣吉尔)、c-IL-2、cat-(X-IL-2)(溶解在PBS溶液中)注入小鼠腹腔。在30分钟、1小时、3小时、6小时、12小时、24小时、48小时和72小时,从眼眶中取出外周血样本(每次200μL)。使用人IL-2ELISA试剂盒(Neobioscience)定量IL-2的浓度,如图3所示,表明拓扑IL-2的半衰期相较于野生型有较明显的提升。
使用链霉亲和素探针进行BLI表征。样品WT组IL-2(欣吉尔)、c-IL-2和cat-(X-IL-2)用缓冲液(PBS、0.05% Tween-20、0.5% BSA)稀释至1000、330、110、37和12nM(IL-2),结果如图4所示,拓扑IL-2与受体的亲和力和野生型的在同一个数量级(IL-2与IL-2Rα和IL-2β结合的KD分别为6.58nM和129nM,参考文献Zhang,B.et al.Site-specific PEGylationof interleukin-2enhances immunosuppression via the sustained activation ofregulatory T cells.Nat.Biomed.Eng.5,1288-1305,doi:10.1038/s41551-021-00797-8(2021))。
实施例3、人外周血PBMC的IL-2刺激实验
筛选系统性红斑狼疮(SLE)、类风湿关节炎(RA)、干燥综合征(SS)患者取外周血,分离人外周血单核细胞(PBMC),用待测样品体外刺激培养72小时,用荧光抗体进行流式染色:anti-CD3-AF700,anti-CD4-FITC,anti-CCR6-BV650,anti-CXCR3-PECF594,anti-CXCR5-AF647,anti-PD-1-PE-cy7,anti-CD3-PerCP,anti-CD127-BV605,anti-CD25-APC。检测细胞分型:Treg(CD3+CD4+CD25+CD127-),Tfh(CD3+CD4+CXCR5+PD-1+),Th1(CD3+CD4+CCR6-CXCR3+),Th2(CD3+CD4+CCR6-CXCR3-)和Th17(CD3+CD4+CCR6+CXCR3-)结果如图5所示,表明在患者中c-IL-2及cat-(X-IL-2)可较高水平地激活Treg细胞,并抑制Tfh及Th1细胞。
实施例4、拓扑IL-2在小鼠体内的活性实验
(1)干燥综合征
购于北京华阜康生物科技股份有限公司的无特定病原体级(specific pathogenfree,SPF)6~8周NOD雌性小鼠,均寄养于北京大学人民医院动物实验室SPF级环境,适应性饲养1周后进行实验。治疗涉及长期给药,WT组(IL-2)一周给药3次,共给药1个月;c-IL-2及cat-(X-IL-2)组一周给药一次,共给药1个月。给药前后给予小鼠15分钟唾液流率测定。给药1个月后取小鼠分离脾脏及淋巴结细胞进行多色流式检测。结果表明,注射c-IL-2及cat-(X-IL-2)可明显促进小鼠脾脏及淋巴结Treg细胞增殖,并且对生发中心B细胞有一定抑制作用,如图6所示。治疗后小鼠的唾液流率表明c-IL-2后小鼠唾液流率有明显升高且与WT组(IL-2)持平,cat-(X-IL-2)组唾液流率有上升趋势但无明显差异(图7)
(2)系统性红斑狼疮
购于北京华阜康生物科技股份有限公司的无特定病原体级(specific pathogenfree,SPF)6~8周C57雌性小鼠,均寄养于北京大学人民医院动物实验室SPF级环境,适应性饲养1周后进行造模实验。造模方法:给予5% IMQ乳膏1.25mg涂抹于小鼠右耳皮肤,每隔1天给药1次,连续给药6周。给药6周末,用酶联免疫吸附实验法检测小鼠血清抗双链DNA(ds-DNA)抗体含量。
治疗涉及长期给药,WT组(IL-2)一周给药3次,共给药2个月;c-IL-2及cat-(X-IL-2)组一周给药一次,共给药2个月。采集小鼠血清进行生化检测,采集肝、肺、心脏、脑等组织进行病理检测。免疫原性是影响蛋白质药物长期给药效果的关键因素之一,因此发明人评价了WT IL-2和c-IL-2、cat-(X-IL-2)在长期给药中的免疫原性。结果表明,c-IL-2及cat-(X-IL-2)具有较高的免疫原性,其诱导小鼠产生淋巴结Treg细胞增殖明显高于WT组,抑制生发中心B细胞增殖作用与WT组持平,如图8所示。证明c-IL-2及cat-(X-IL-2)在长期给药中具有较高的免疫原性。对小鼠模型HE染色,进一步发现c-IL-2和cat-(X-IL-2)炎症聚集明显低于空白组(PBS),且与WT组(IL-2)差别不大(图9)。此外,治疗后,发现抗核抗体(ANA)含量在注射c-IL-2及cat-(X-IL-2)后的小鼠中明显低于空白组(PBS),如图10所示。图11还进一步展示了治疗后小鼠尿蛋白比WT组明显下降。图12显示,拓扑IL-2并无明显生理毒性。
(3)类风湿关节炎
购于北京华阜康生物科技股份有限公司的无特定病原体级(specific pathogenfree,SPF)6~8周DBA/1雄性小鼠,均寄养于北京大学人民医院动物实验室SPF级环境,适应性饲养1周后进行造模实验。造模方式:牛II型胶原(Bovine Type II Collagen,C II)用100mmol/L冰醋酸4℃过夜溶解,与完全弗氏佐剂(Complete Freund’s Adjuvant,CFA)按照1∶1的体积比混合,充分乳化。在小鼠尾根部分两点进行皮下注射,各注射含100μg C II的乳化液;初次免疫后第21天,C II和不完全弗氏佐剂(Incomplete Freund’s Adjuvant,IFA)按照1∶1的体积比充分乳化,在小鼠尾根部皮下注射含100μg C II的乳化液,完成胶原诱导性关节炎(CIA)模型的诱导。二次免疫后6天,小鼠陆续发病,14天达到高峰期,即获得CIA小鼠。
治疗涉及长期给药,WT组(IL-2)一周给药3次,共给药2个月;c-IL-2及cat-(X-IL-2)组一周给药一次,共给药2个月。隔天对小鼠关节炎严重情况进行评分:0=无红肿;1=小趾关节肿胀;2=趾关节和组织部肿胀;3=踝关节以下的足爪肿胀;4=包括踝关节在内的全部足爪肿胀(图13)。给药两个月后取小鼠分离脾脏及淋巴结细胞进行多色流式检测。如图14所示,结果表明,注射c-IL-2及cat-(X-IL-2)的实验组小鼠淋巴结Treg细胞显著高于PBS组,且略高于WT组;脾脏Treg细胞也显著高于PBS组。注射c-IL-2及cat-(X-IL-2)的实验组小鼠淋巴结Th1、Th2、Th17细胞显著低于PBS组,c-IL-2及cat-(X-IL-2)的实验组小鼠淋巴结Th2细胞显著低于WT组小鼠,并且可以有效地防治关节炎的发生发展。
Claims (9)
1.一种白细胞介素-2拓扑改造蛋白,为环状白细胞介素-2或索烃白细胞介素-2。
2.如权利要求1所述的白细胞介素-2拓扑改造蛋白,其特征在于,所述环状白细胞介素-2是将白细胞介素-2的N端和C端相连所形成的环状结构的白细胞介素-2;所述索烃白细胞介素-2是将白细胞介素-2与能够形成二聚体的蛋白质缠结基元融合表达,通过蛋白质缠结基元形成分子间的缠结二聚体并形成闭环结构,得到具有索烃结构的白细胞介素-2二聚体。
3.如权利要求2所述的白细胞介素-2拓扑改造蛋白,其特征在于,所述环状白细胞介素-2的氨基酸序列如序列表中SEQ ID No:1所示。
4.如权利要求2所述的白细胞介素-2拓扑改造蛋白,其特征在于,所述蛋白质缠结基元是肿瘤抑制因子p53dim。
5.如权利要求4所述的白细胞介素-2拓扑改造蛋白,其特征在于,所述索烃白细胞介素-2由两个相同的环构成,每个环的氨基酸序列如序列表中SEQ ID No:2所示。
6.权利要求1~5任意一项所述的白细胞介素-2拓扑改造蛋白在制备治疗免疫相关疾病的药物中的应用。
7.如权利要求6所述的应用,其特征在于,所述免疫相关疾病包括自身免疫性疾病、免疫缺陷病、肿瘤、病毒感染。
8.如权利要求7所述的应用,其特征在于,所述自身免疫性疾病包括系统性红斑狼疮、类风湿关节炎、干燥综合征、脏器移植排斥反应、移植物抗宿主疾病、皮肌炎、多发性硬化。
9.一种药物组合物,包含权利要求1~5任意一项所述的白细胞介素-2拓扑改造蛋白。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013184942A1 (en) * | 2012-06-08 | 2013-12-12 | Alkermes, Inc. | Ligands modified by circular permutation as agonists and antagonists |
CN105061581A (zh) * | 2015-09-17 | 2015-11-18 | 北京大学 | 可基因编码的全蛋白质索烃的制备方法 |
CN111560391A (zh) * | 2020-05-21 | 2020-08-21 | 北京大学 | 一种蛋白质异质索烃的生物合成方法 |
CN114555126A (zh) * | 2019-06-11 | 2022-05-27 | 奥克梅斯制药爱尔兰有限公司 | 用于皮下施用癌症免疫疗法的组合物和方法 |
-
2023
- 2023-07-07 CN CN202310827760.9A patent/CN117003852A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013184942A1 (en) * | 2012-06-08 | 2013-12-12 | Alkermes, Inc. | Ligands modified by circular permutation as agonists and antagonists |
CN105061581A (zh) * | 2015-09-17 | 2015-11-18 | 北京大学 | 可基因编码的全蛋白质索烃的制备方法 |
CN114555126A (zh) * | 2019-06-11 | 2022-05-27 | 奥克梅斯制药爱尔兰有限公司 | 用于皮下施用癌症免疫疗法的组合物和方法 |
CN111560391A (zh) * | 2020-05-21 | 2020-08-21 | 北京大学 | 一种蛋白质异质索烃的生物合成方法 |
Non-Patent Citations (1)
Title |
---|
DONG LIU等: "Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability", ACS CENT SCI ., vol. 3, no. 5, pages 473 - 481 * |
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