CN113403276A - 抗体功能化的外泌体制剂及其制备方法和应用 - Google Patents
抗体功能化的外泌体制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种抗体功能化的外泌体制剂及其制备方法和应用。所述外泌体制剂是以特定的肿瘤抗原Ag激活的树突状细胞的外泌体Exo‑Ag为载体,利用磷脂‑聚乙二醇‑琥珀酰亚胺酯DSPE‑PEG‑NHS为交联剂,将抗CD3抗体aCD3和抗EGFR抗体aEGFR修饰到外泌体Exo‑Ag表面而得到的外泌体制剂Exo‑Ag‑aCD3/aEGFR。本发明利用功能化的树突细胞外泌体作为中间体,经过肿瘤特异性抗原活化、具有靶向性的CAR‑T细胞模拟物的构建,克服了CAR‑T疗法中因肿瘤表面抗原的缺失导致的抗性作用,解决了CAR‑T疗法中因体外改造、扩增T细胞造成的时间及成本问题。
Description
技术领域
本发明涉及肿瘤免疫治疗药物技术领域,具体地说是一种抗体功能化的外泌体制剂及其制备方法和应用。
背景技术
CAR-T细胞疗法又叫嵌合抗原受体T细胞免疫疗法,它属于过继性T细胞转移的一种。CAR-T细胞疗法作为一种治疗肿瘤的新型精准靶向疗法,它能够精准、快速、高效,且有可能治愈癌症,是一种极具前景的新型肿瘤免疫治疗方法。然而,广泛的研究揭示了CAR-T细胞免疫疗法中存在的局限性。
CAR-T细胞疗法的原理是利用病人自身的免疫细胞实现对癌细胞的清除,其主要目的是加强免疫系统内在的抗癌能力。CAR-T细胞疗法是从患者的血液中提取T细胞,并在体外对这些细胞进行基因改造,给它们装上识别癌细胞表面抗原的“嵌合抗原受体”(CAR)。随后,将这些改造之后的细胞进行大量扩增,再输注回患者体内,从而达到识别、杀死癌细胞的治疗效果。CAR-T细胞的嵌合抗原受体(CAR)是由胞外抗原结合域、铰链区、跨膜区和胞内结构域组成的。当把重新编码后的T细胞回输进患者体内后,这种嵌合抗原受体就像“定位导航装置”一样可以特异性地追踪和识别并引导T细胞杀伤肿瘤细胞。
对目前来说,CAR-T细胞发展仍未完善,仍有诸多问题有待解决。首先,肿瘤表面抗原的缺失导致的抗性作用以及CAR-T的功能持久性较差,仍可能导致疾病进展。其次,大剂量CAR-T细胞输注后可能会引发细胞因子释放综合征等严重的副作用,这也是CAR-T技术在临床应用中一个最主要的不良反应。此外,体外改造、扩增并回输患者T细胞的操作非常费力且费用高也是CAR-T疗法的应用限制之一。且由于不同患者T细胞反应、持续性和副作用的不同,预测最佳输注细胞数也是一个挑战。更值得注意的是,由于肿瘤促结缔组织增生的特性和免疫抑制的肿瘤微环境使得CAR-T细胞通常在肿瘤中的浸润效率较低,以至于CAR-T细胞疗法对于实体瘤的治疗效果并不理想。因此,能够克服CAR-T细胞疗法中的应用障碍的设计策略对于增强其临床应用价值具有重要意义。
发明内容
本发明的目的就是提供一种抗体功能化的外泌体制剂及其制备方法和应用,以解决现有CAR-T细胞疗法存在的体外改造扩增T细胞操作繁琐、储存/运输稳定性低、脱靶、保留率低等的问题。
本发明的目的是这样实现的:一种抗体功能化的外泌体制剂,所述外泌体制剂是以特定的肿瘤抗原Ag激活的树突状细胞的外泌体Exo-Ag为载体,利用磷脂-聚乙二醇-琥珀酰亚胺酯DSPE-PEG-NHS为交联剂,将抗CD3抗体aCD3和抗EGFR抗体aEGFR修饰到外泌体Exo-Ag表面而得到的外泌体制剂Exo-Ag-aCD3/aEGFR。
所述树突状细胞为骨髓来源的树突状细胞、DC2.4细胞系、外周血树突状细胞中的至少一种。
所述特定的肿瘤抗原Ag为OVA抗原、黑色素瘤相关抗原 MAGE以及通过细胞或组织破碎提取出的黑色素瘤特异性抗原中的至少一种。
上述的外泌体制剂的制备方法,包括以下步骤:
(a)培养树突状细胞:将保存的树突状细胞进行活化并接种于培养皿中,在含5%CO2的37℃细胞培养箱中培养;
(b)提取外泌体:将特定的肿瘤抗原Ag与树突状细胞共孵育,孵育完成后,收集细胞培养上清液,利用梯度离心法,除去细胞碎片和大颗粒蛋白杂质,之后利用超滤离心管进行浓缩除杂,得到外泌体溶液Exo-Ag;
(c)抗体修饰Exo-Ag:将磷脂-聚乙二醇-琥珀酰亚胺酯DSPE-PEG-NHS溶于DMSO中,将经洗涤后的抗体aCD3和抗体aEGFR分别与DSPE-PEG-NHS溶液混合,其中,抗体aCD3或抗体aEGFR 与DSPE-PEG-NHS的摩尔比为1∶1-3,混合后在2-4℃条件下持续搅拌反应20-28h;反应完成后离心除去未反应的DSPE-PEG-NHS,分别得到DSPE-PEG-NHS-aCD3溶液和DSPE-PEG-NHS-aEGFR溶液;将DSPE-PEG-NHS-aCD3溶液和DSPE-PEG-NHS-aEGFR溶液与Exo-Ag于 2-4℃条件下反应2-4h,使DSPE-PEG-NHS-aCD3和DSPE-PEG-NHS-aEGFR的DSPE端插入到外泌体的膜磷脂双分子层中,从而得到成功修饰aCD3和aEGFR的外泌体制剂Exo-Ag-aCD3/aEGFR。
步骤(a)中,将冻存的树突状细胞取出,在37℃水浴锅内使其融化,然后移入EP管中,加入预热的1640完全培养基,吹匀后进行离心,弃上清液,再加入1640完全培养基,并吹匀后接种于培养皿中进行培养。
步骤(b)中,将0.1-0.5 mg/mL 的肿瘤抗原Ag与树突状细胞共孵育12-24h后,更换新鲜培养基48 h后收集细胞培养上清液进行过滤。
步骤(c)中,利用10 KDa超滤离心管除去过量的DSPE-PEG-NHS,随后将抗体aCD3和抗体aEGFR的溶液超速离心除去溶剂,并用PBS洗涤并溶解后回收备用。
上述的外泌体制剂在靶向抗肿瘤药物中的应用。
所述药物为注射剂,所述药物包含外泌体制剂Exo-Ag-aCD3/aEGFR。
本发明根据CAR-T细胞的胞内、外结构域的作用,利用功能化的树突细胞外泌体作为中间体,经过肿瘤特异性抗原活化、具有靶向性的CAR-T细胞模拟物的构建,克服了CAR-T疗法中因肿瘤表面抗原的缺失导致的抗性作用,解决了CAR-T疗法中因体外改造、扩增T细胞造成的时间及成本问题。
本发明联合aCD3和aEGFR,通过具有双靶点的外泌体作为中间桥梁,增强T细胞向肿瘤组织的浸润以及向癌细胞表面的锚定。实验证明,将Exo-OVA-aCD3/aEGFR(OVA为一种广泛使用的抗原模型)尾静脉注射给荷瘤小鼠后,基于DC 2.4外泌体的归巢作用,Exo-OVA-aCD3/aEGFR聚集至淋巴组织中并附着于T细胞表面。Exo-OVA-aCD3/aEGFR表面的MHC II分子将携带的抗原肽呈递给T细胞进而刺激T细胞活化。随后,在血液循环过程中,经活化的T细胞在aEGFR靶向性的双重作用下向肿瘤组织浸润并最终锚定在癌细胞上以释放细胞毒素杀伤癌细胞。MHC II分子充当CAR-T细胞的胞内结构域,通过呈递OVA抗原促进T细胞的活化,而aCD3和aEGFR的组合使用充当了CAR-T细胞的胞外结构域作为T细胞的 “定位导航装置”。通过这种方式,实现了经过肿瘤特异性抗原活化的、具有靶向性的CAR-T细胞模拟物在体内的构建及显著的免疫治疗效果。
本发明制备方法简单,通过特定的交联剂得到aCD3和aEGFR修饰的Exo-Ag,降低了制备成本,减少了毒副作用。
附图说明
图1是本发明Exo-OVA-aCD3/aEGFR的制备原理图和传统CAR-T与无细胞CAR-T的对比示意图。
图2是Exo-OVA的电镜图。
图3是Exo-OVA-aCD3/aEGFR合成过程中的凝胶电泳图。
图4是Exo-OVA-aCD3/aEGFR的蛋白质印迹分析图。
图5是Exo-OVA-aCD3/aEGFR的纳米流式仪分析图。
图6是Exo-OVA-aCD3/aEGFR的粒径分布图。
图7是流式细胞仪分析Exo-OVA-aCD3/aEGFR体外免疫激活能力。
图8是共聚焦验证Exo-OVA-aCD3/aEGFR增强T细胞向癌细胞表面锚定作用。
图9是Exo-OVA-aCD3/aEGFR对肿瘤生长的抑制作用。
图10是Exo-OVA-aCD3/aEGFR治疗后荷瘤小鼠的存活率。
图11是器官及肿瘤组织的病理分析。
图12是Exo-OVA-aCD3/aEGFR治疗后流式细胞仪分析肿瘤局部T细胞浸润情况。
图13是治疗后小鼠血清中的相关免疫细胞因子的测定。
图14是小鼠肿瘤复发情况测定。
图15是荷瘤小鼠肺组织转移结节测定。
具体实施方式
下面结合具体实施例对本发明的技术方案进行详细说明。本发明实施例中未提到的试验条件和操作均按本领域常规方法或制造商建议的条件进行。
实施例1:
下面以一种广泛使用的抗原模型OVA为例,详细介绍Exo-OVA-aCD3/aEGFR的制备方法,如图1所示,包括如下步骤:
(1)培养树突状细胞(DC 2.4)
将冻存的DC 2.4细胞从液氮中取出,在37 ℃水浴锅内不断摇动促进其融化。移入10 mL EP管中,加入10 mL预热的1640完全培养基,轻轻吹匀,2000 rpm离心5 min,弃上清液。加入10 mL 1640完全培养基,轻轻吹打,接种于培养皿中,在含5% CO2的37 ℃细胞培养箱中培养。
(2)提取外泌体
0.1 mg/mL OVA与DC 2.4共孵育24 h后,更换新鲜培养基48 h后收集细胞培养上清液,利用梯度离心法,除去细胞碎片及大颗粒蛋白等杂质。随后利用100 KD超滤离心管在4000 g转速下离心5 min,浓缩的同时除去培养液中的小颗粒杂质。浓缩后得到纯度较好的外泌体溶液(Exo-OVA)。可以于-80°C冰箱中长期保存,或在4°C冰箱中至多可保存一周。
(3)抗CD3抗体和抗EGFR抗体修饰Exo-OVA
Exo-OVA分离纯化后利用抗CD3抗体(aCD3)和抗EGFR抗体(aEGFR)进行表面修饰。为防止aCD3和aEGFR的溶剂干扰抗体与连接剂的结合反应,利用10 KDa超滤离心管在2000g,5 min条件下离心除去溶剂,PBS洗涤一次,回收备用。选用磷脂-聚乙二醇-琥珀酰亚胺酯(DSPE-PEG-NHS)为交联剂。将DSPE-PEG-NHS溶于DMSO,与离心后的aCD3和aEGFR分别以2:1比例混合,在4 °C条件下持续搅拌反应24 h,aCD3和aEGFR与DSPE-PEG-NHS通过酰胺化反应连接。4500 g超滤离心5 min,除去未反应的DSPE-PEG-NHS,上层溶液DSPE-PEG-NHS-aCD3或DSPE-PEG-NHS-aEGFR回收后与纯化后的Exo-OVA在 4 °C条件下反应3 h,DSPE-PEG-NHS-aCD3或DSPE-PEG-NHS-aEGFR的DSPE端插入外泌体的膜磷脂双分子层中,成功修饰aCD3和aEGFR得到Exo-OVA-aCD3/aEGFR。
Exo-OVA-aCD3/aEGFR的制备原理如图1A所示,传统CAR-T与无细胞CAR-T的对比原理如图1B所示。对Exo-OVA-aCD3/aEGFR进行表征,结果如图2-图6所示。
图2通过生物电镜观察,提取的DC 2.4细胞的外泌体(Exo-OVA)为100nm左右的球形中空囊泡。
图3凝胶电泳结果显示aCD3、aEGFR与DSPE-PEG-NHS键连后(Exo-OVA-aCD3、Exo-OVA-aEGFR)电泳条带位于纯aCD3、aEGFR条带之上,说明aCD3、aEGFR与连接剂键连后分子量变大,且条带弥散,证明aCD3、aEGFR与DSPE-PEG-NHS成功通过酰胺化反应连接。
图4 Exo-OVA-aCD3/aEGFR的Western blot定性分析结果表明,提取的囊泡表达CD63、CD81等标记蛋白,证实所提取的细胞外囊泡属于外泌体。且OVA刺激后DC 2.4分泌的外泌体成功表达OVA抗原,表示Exo-OVA-aCD3/aEGFR具有呈递抗原活化T细胞的功能。
图5纳米流式仪结果显示,与对照组相比,Exo-OVA-aCD3/aEGFR组的外泌体检测到了荧光抗体(aCD3-PE/Cy5、aEGFR-FITC)的信号,也证明了aCD3、aEGFR被修饰到了外泌体表面。
图6 纳米颗粒追踪分析仪(NTA)测定Exo-OVA-aCD3/aEGFR粒径为102.9 nm,浓度为7.3×107个/mL。
实施例2
对Exo-OVA-aCD3/aEGFR的抗肿瘤效果进行研究,结果如下:
(1)Exo-OVA-aCD3/aEGFR体外活化T细胞能力评价
通过检测T细胞的分化情况,验证Exo-OVA-aCD3/aEGFR的体外活化T细胞的能力。将T细胞与Exo、Exo-OVA、Exo-OVA-aCD3/aEGFR孵育后,离心PBS重悬细胞并用荧光抗体CD4、CD8标记T细胞。用流式细胞术检测,结果如图7所示,Exo与对照组基本相同,没有活化T细胞的能力。而Exo-OVA、Exo-OVA-aCD3/aEGFR均能不同程度地激活T细胞,促进其增殖分化。体外T细胞分化实验表明,携带OVA抗原的外泌体能够有效促进T细胞的活化。
(2)Exo-OVA-aCD3/aEGFR增强T细胞向癌细胞锚定的能力评价
通过共聚焦观察,验证Exo-OVA-aCD3/aEGFR增强T细胞向癌细胞锚定的能力。将DiO染色的T细胞与Exo-OVA-aCD3、Exo-OVA-aEGFR、Exo-OVA-aCD3/aEGFR孵育30 min后,加入DiD染色的B16-OVA细胞共孵育1 h。通过共聚焦观察Exo-OVA-aCD3/aEGFR介导的T细胞与B16-OVA细胞的交联,结果如图8所示,相比于Exo-OVA-aCD3、Exo-OVA- aEGFR,Exo-OVA-aCD3/aEGFR能有效的介导T细胞向癌细胞的锚定。
(3)Exo-OVA-aCD3/aEGFR体内抑瘤水平评价
为了验证利用Exo-OVA-aCD3/aEGFR在体内构建CAR-T样免疫治疗平台是否能增强抑制肿瘤生长的作用,将不同材料PBS(G1)、Exo(G2)、Exo-OVA(G3)、Exo-OVA-aCD3(G4)、Exo-OVA-aEGFR(G5)、Exo-OVA-aCD3/aEGFR(G6)(Exo 1010/只,aPD-L1 40 µg/只)尾静脉给药B16-OVA荷瘤小鼠。同时每两天测量各组小鼠的肿瘤大小,检测肿瘤生长情况。如图9,Exo-OVA-aCD3/aEGFR治疗组对肿瘤生长的抑制作用最显著,与PBS对照组相比有极显著的差异性。而Exo、Exo-OVA、Exo-OVA-aCD3、Exo-OVA-aEGFR等治疗组对肿瘤生长的抑制作用有限,其中Exo-OVA-aCD3/aEGFR组与Exo-OVA、Exo-OVA-aCD3、Exo-OVA-aEGFR组肿瘤体积存在显著差异。说明aCD3和aEGFR的组合使用增加T细胞向癌细胞表面的锚定,有助于肿瘤的免疫治疗。此外,直到治疗后40天,Exo-OVA-aCD3/aEGFR治疗组的肿瘤体积生长速度仍然较慢,显示出明显的抗肿瘤作用。
图10结果表明,Exo-OVA-aCD3/aEGFR的治疗有效地延长了小鼠的平均存活时间,直至40天实验结束仍旧有6只小鼠存活。
图11表明H&E染色的心、肝、脾、肺、肾,观察各组器官并无明显损伤。肿瘤组织染色结果显示经Exo-OVA-aCD3/aEGFR处理的肿瘤组织明显坏死。
治疗21天后,收集荷瘤小鼠的肿瘤样本利用流式细胞术进行分析。如图12所示,与未处理组相比,Exo-OVA-aCD3/aEGFR组CD4 + T细胞和CD8+ T细胞的浸润明显增加。值得注意的是,Exo-OVA-aCD3/aEGFR组分比Exo-OVA、Exo-OVA-aCD3、Exo-OVA-aEGFR组更有利于免疫细胞的浸润,这是由于Exo-OVA-aCD3/aEGFR通过aCD3结合在T细胞表面后aEGFR可以作为T细胞的靶分子增加其向癌细胞表面的锚定所致。肿瘤局部免疫细胞的浸润增加,可有效改善肿瘤局部的免疫抑制性微环境,显著增强肿瘤免疫治疗效果。已有研究表明肿瘤浸润性淋巴细胞(TIL)的比例提高可有效逆转抑制性微环境。
治疗过程中对血清中的相关免疫细胞因子进行分析。如图13 所示,细胞因子分泌的增加,包括干扰素-γ、肿瘤坏死因子-α和白细胞介素-6等,进一步证实了Exo-OVA-aCD3/aEGFR能有效地激活天然免疫和获得性免疫反应,产生有效杀伤。
(4)Exo-OVA-aCD3/aEGFR抑制肿瘤转移复发效果评价
为了测试Exo-OVA-aCD3/aEGFR的治疗潜力,建立复发转移小鼠模型进行了试验。基于小鼠手术后肿瘤复发体积,相比于对照组,Exo-OVA-aCD3/aEGFR治疗显著有效的阻止了局部肿瘤的复发,如图14显示。
小鼠肿瘤复发转移模型中,收集转移肺组织,分析每组肺组织统计肺结节数量,发现空白组有明显的转移点,而经过Exo-OVA-aCD3/aEGFR治疗的小组,肺转移的平均数量显著减少,与空白组存在显著性差异,结果可见图15。
Claims (9)
1.一种抗体功能化的外泌体制剂,其特征是,所述外泌体制剂是以特定的肿瘤抗原Ag激活的树突状细胞的外泌体Exo-Ag为载体,利用磷脂-聚乙二醇-琥珀酰亚胺酯DSPE-PEG-NHS为交联剂,将抗CD3抗体aCD3和抗EGFR抗体aEGFR修饰到外泌体Exo-Ag表面而得到的外泌体制剂Exo-Ag-aCD3/aEGFR。
2.根据权利要求1所述的外泌体制剂,其特征是,所述树突状细胞为骨髓来源的树突状细胞、DC2.4细胞系、外周血树突状细胞中的至少一种。
3.根据权利要求1所述的外泌体制剂,其特征是,所述特定的肿瘤抗原Ag为OVA抗原、黑色素瘤相关抗原 MAGE以及通过细胞或组织破碎提取出的黑色素瘤特异性抗原中的至少一种。
4.权利要求1所述的外泌体制剂的制备方法,其特征是,包括以下步骤:
(a)培养树突状细胞:将保存的树突状细胞进行活化并接种于培养皿中,在含5% CO2的37℃细胞培养箱中培养;
(b)提取外泌体:将特定的肿瘤抗原Ag与树突状细胞共孵育,孵育完成后,收集细胞培养上清液,利用梯度离心法,除去细胞碎片和大颗粒蛋白杂质,之后利用超滤离心管进行浓缩除杂,得到外泌体溶液Exo-Ag;
(c)抗体修饰Exo-Ag:将磷脂-聚乙二醇-琥珀酰亚胺酯DSPE-PEG-NHS溶于DMSO中,将经洗涤后的抗体aCD3和抗体aEGFR分别与DSPE-PEG-NHS溶液混合,其中,抗体aCD3或抗体aEGFR与DSPE-PEG-NHS的摩尔比为1∶1-3,混合后在2-4℃条件下持续搅拌反应20-28h;反应完成后离心除去未反应的DSPE-PEG-NHS,分别得到DSPE-PEG-NHS-aCD3溶液和DSPE-PEG-NHS-aEGFR溶液;将DSPE-PEG-NHS-aCD3溶液和DSPE-PEG-NHS-aEGFR溶液与Exo-Ag于 2-4℃条件下反应2-4h,使DSPE-PEG-NHS-aCD3和DSPE-PEG-NHS-aEGFR的DSPE端插入到外泌体的膜磷脂双分子层中,从而得到成功修饰aCD3和aEGFR的外泌体制剂Exo-Ag-aCD3/aEGFR。
5.根据权利要求4所述的制备方法,其特征是,步骤(a)中,将冻存的树突状细胞取出,在37℃水浴锅内使其融化,然后移入EP管中,加入预热的1640完全培养基,吹匀后进行离心,弃上清液,再加入1640完全培养基,并吹匀后接种于培养皿中进行培养。
6.根据权利要求4所述的制备方法,步骤(b)中,将0.1-0.5 mg/mL 的肿瘤抗原Ag与树突状细胞共孵育12-24h后,更换新鲜培养基48 h后收集细胞培养上清液进行过滤。
7.根据权利要求4所述的制备方法,其特征是,步骤(c)中,利用10 KDa超滤离心管除去过量的DSPE-PEG-NHS,随后将抗体aCD3和抗体aEGFR的溶液超速离心除去溶剂,并用PBS洗涤并溶解后回收备用。
8.权利要求1-3任一所述的外泌体制剂在靶向抗肿瘤药物中的应用。
9.根据权利要求8所述的应用,其特征是,所述药物为注射剂,所述药物包含外泌体制剂Exo-Ag-aCD3/aEGFR。
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