CN115006550A - 偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体 - Google Patents
偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体 Download PDFInfo
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Abstract
本发明属于医药技术领域,涉及一种偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体及其在治疗恶性实体肿瘤中的用途,该肿瘤治疗外泌体能够触发T细胞介导的抗肿瘤免疫应答,从而抑制肿瘤血液途径的转移,消融原发肿瘤,有效避免术后肿瘤复发,并引发了机体的长期免疫记忆。所述的肿瘤治疗外泌体是以特定的肿瘤抗原激活、特定基因编码的重组腺病毒转染的树突状细胞外泌体为载体,以生物素‑链霉亲和素‑生物素复合物为连接臂,将aCTLA4抗体修饰到外泌体表面而得的aCTLA4修饰的肿瘤治疗外泌体。本发明的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体可以用于制备治疗抗肿瘤或抗肿瘤转移的药物。
Description
技术领域
本发明属于医药技术领域,涉及一种偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体及其在治疗恶性实体肿瘤中的用途,该肿瘤治疗外泌体能够触发T细胞介导的抗肿瘤免疫应答,从而抑制肿瘤血液途径的转移,消融原发肿瘤,有效避免术后肿瘤复发,并引发了机体的长期免疫记忆。
背景技术
树突状细胞(DC)是免疫系统的总司令。DC衍生的外泌体(Dex)的分子组成与DC非常相似,包括共刺激分子、主要组织相容性复合物(MHC)/抗原肽以及白介素(IL)-15受体α(IL-15Rα)的表面表达。不含细胞的Dex避免了病毒或细胞疗法有关的风险,包括体内复制的风险,Dex可在淋巴结中广泛分布并且可以通过膜表面的受体接触各类免疫细胞。Dex可以以间接和直接方式刺激T细胞成熟。初始T细胞的活化是通过DC间接呈递Dex上的抗原肽而发生的;同时Dex可以直接刺激活化的T细胞并触发其增殖。但是,目前临床试验中的Dex免疫应答不令人满意,基于工程化的Dex的免疫疗法为解决这一问题带来了希望。
呈递内源性抗原是引发适应性免疫反应的有效方法,并且可以诱导免疫记忆。但是对于许多肿瘤,大多数内源性肿瘤抗原仍是未知的。热休克蛋白70(HSP70)在多种肿瘤中过表达,已被证明是可以产生肿瘤抗原的某些多肽的伴侣蛋白,可以被DC呈递并激活CD8+T细胞免疫应答。高免疫原性使HSP70伴侣多肽(HCP)在个性化肿瘤免疫治疗中具有重要作用。
IL-15在引发体内抗肿瘤免疫反应的过程中起着关键作用。它是一种膜相关的生长因子,可激活T细胞分裂,防止T细胞死亡并有助于记忆T细胞的生成和存活。研究表明,IL-15通过与由IL-15受体(IL-15R)α和IL-15Rβ/γ组成的高亲和力三聚体复合物结合而引发生物学效应。IL-15与DC膜上的IL-15Rα结合,并将其反式呈递给膜上表达IL-15Rβ/γ的相邻T细胞。IL-15Rα被证明保留在Dex的膜上。因此,我们希望借助膜锚定的IL-15增强Dex诱导的抗肿瘤免疫。
只有抗原的呈递无法有效激活T细胞,除了T细胞受体(TCR)与MHC/抗原肽的相互作用外,共刺激信号对于T细胞免疫的启动也是必不可少的。T细胞表面的CD28和抗原呈递细胞(APC)表面的配体CD80、CD86是最重要的共刺激信号。细胞毒性T淋巴细胞相关蛋白4(CTLA4)是CD28的同源物,它以比CD28高得多的亲和力结合CD80和CD86。但是,CTLA4介导的是免疫抑制信号,会导致活化T细胞的凋亡。阻断CTLA4介导的免疫抑制信号可增强T细胞对癌细胞的反应并改善免疫治疗效果。
发明内容
本发明所解决的技术问题是为了克服现有技术的缺陷,设计了偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体,即通过生物素-链霉亲和素-生物素复合物将抗CTLA4抗体(aCTLA4)结合到工程化的Dex表面,从而实现了强大的非共价结合相互作用并确保了其在复杂生理环境中的高稳定性。内源性HCP刺激基因工程化IL-15/IL-15Rα过表达的DC。分离提取工程化的Dex并用aCTLA4的修饰,Dex保留了膜结合的MHC/抗原肽复合物和IL-15/IL-15Rα复合物。系统给药后,由于aCTLA4与CTLA4的特异性识别能力,高度免疫原性的肿瘤自身抗原呈递以及IL-15反式呈递的协同作用,该肿瘤治疗外泌体表现出对淋巴组织增强的归巢和改善的免疫反应。在不同小鼠肿瘤模型中表现出了增强的T细胞反应,显著抑制了肿瘤的转移和术后复发,产生了长期的免疫记忆。
具体地,本发明的第一个目的是,克服传统肿瘤治疗外泌体治疗恶性实体肿瘤效果不佳的缺点,提供一种基因工程化外泌体偶联免疫检查点阻断单克隆抗体的新型肿瘤治疗外泌体。
本发明的第二个目的是,提供所述偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体的制备方法。
本发明的第三个目的是,提供所述偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体在制备抗肿瘤治疗外泌体中的应用。
为实现上述第一个目的,本发明采取的技术方案是:提供偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体,其特征在于,所述的肿瘤治疗外泌体是以特定的肿瘤抗原激活、特定基因编码的重组腺病毒转染的树突状细胞外泌体为载体,以生物素-链霉亲和素-生物素复合物为连接臂,将aCTLA4抗体修饰到外泌体表面而得的aCTLA4修饰的肿瘤治疗外泌体。
所述的树突状细胞为骨髓来源的树突状细胞、外周血树突状细胞、DC2.4中的至少一种。
所述的特定肿瘤抗原为HSP70伴侣多肽、OVA、MAGE以及通过细胞或组织破碎提取出来的肿瘤特异性抗原中的至少一种。
所述的特定基因为IL-15、IL-15Rα中的至少一种。
所述的生物素-链霉亲和素-生物素复合物为sulfo-NHS-生物素-链霉亲和素-aCTLA4-生物素。
进一步地,所述的树突状细胞外泌体、特定基因、aCTLA4抗体的质量比为1-2:1-2:80-130,优选为1.5-1.8:1.4-1.6:100-110,更优选为1.8:1.4:100。
本发明优选以HSP70伴侣多肽为肿瘤抗原、IL-15编码的重组腺病毒转染的树突状细胞外泌体为载体,以生物素-链霉亲和素-生物素复合物为连接臂,将aCTLA4抗体修饰到外泌体表面而得的aCTLA4修饰的肿瘤治疗外泌体。
所述的树突状细胞外泌体、IL-15、aCTLA4的质量比为1-2:1-2:80-130,优选为1.5-1.8:1.4-1.6:100-110,更优选为1.8:1.4:100。
为实现上述第二个目的,所述的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体的制备步骤包含IL-15/IL-15Rα基因编码的重组腺病毒转染树突状细胞、肿瘤抗原刺激树突状细胞成熟、免疫检查点阻断单克隆抗体修饰提取的树突状细胞外泌体。
其具体步骤如下:
(1)培养树突状细胞,将骨髓细胞均匀分散在细胞培养板中培养,使用GM-CSF和IL-4诱导骨髓细胞分化为树突状细胞。
(2)提取外泌体:用特定基因编码的重组腺病毒转染树突状细胞,并用特定抗原刺激树突状细胞成熟,收集细胞培养上清,经过超速离心法提取外泌体。
(3)aCTLA4抗体修饰外泌体:为了获得aCTLA4-生物素,将aCTLA4与sulfo-NHS(N-羟基硫代琥珀酰亚胺)-生物素在PBS中混合,并在室温下轻轻摇动反应0.5-1小时。使用超滤管纯化aCTLA4-生物素,在1600rcf下离心3-6分钟。将外泌体分别与sulfo-NHS-生物素,链霉亲和素和aCTLA4-生物素反应1-2小时。并在PBS中洗涤三次,以除去每次反应间的游离物。
步骤(1)中,在细胞培养板中加入含有10%胎牛血清、10-30ng/ml的GM-CSF和10-30ng/ml的IL-4的RPMI1640培养基,每隔两天用含有20-60ng/ml的GM-CSF和20-60ng/ml的IL-4的新鲜培养基半量换液。
步骤(2)中,用特定基因编码的重组腺病毒转染树突状细胞48小时后,用含有0.2-0.6mg/ml的肿瘤抗原与树突状细胞孵育12-24小时后,更换含有无外泌体血清的细胞培养液继续培养24-48小时并收集上清液提取外泌体。
步骤(3)中,利用10kDa超滤离心管将aCTLA4-生物素溶液离心除去游离的生物素。
为实现上述第三个目的,本发明制备了偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex),并对其性质进行了考察,同时制备了树突状细胞外泌体(Dex),外泌体偶联免疫检查点阻断单克隆抗体(aCTLA4-Dex)以及基因工程化外泌体(IL-15-Dex)作为对照,详细考察内容如下:
(1)制备了偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex),对其理化性质进行表征,如粒径、形态、Western Blot表征蛋白表达、修饰效率等。
(2)考察偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的体外T细胞活化能力及T细胞靶向和体内分布。
(3)考察偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)在尾静脉注射肿瘤细胞的Balb/c小鼠体内的抗转移治疗效果。
(4)考察偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)在右侧第四对乳腺脂肪垫上接瘤的Balb/c小鼠术后肿瘤模型的抗复发治疗效果。
(5)考察偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)在经治疗的Balb/c小鼠荷瘤模型重新接种肿瘤后的记忆性免疫保护作用。
结果表明,本发明制备的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体抑制了肿瘤血液途径的转移,消融了原发肿瘤,有效避免了术后肿瘤复发,并引发了机体的长期免疫记忆。
附图说明
图1为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的制备示意图。
图2为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的表征:
A:通过免疫组化比较了4T1和CT26肿瘤组织和正常组织之间HSP70表达的差异。
B:通过DLS测量了提取的HCP的粒径。
C:分离的HCP和4T1,CT26肿瘤细胞的SDS-PAGE蛋白分析。
D:IL-15-Dex的透射电子显微镜图像。右下:MHC II(10nm金颗粒)的免疫胶体金标记。比例尺:100nm。
E:通过NTA测量的IL-15-Dex的粒径分布。
F:IL-15-DC和IL-15-Dex上外泌体相关蛋白(TSG101,CD9和CD81)和DC相关蛋白(MHC I,MHC II和CD86)的Western Blotting分析。
G:aCTLA4/IL-15-Dex上IL-15和aCTLA4的CLSM图像。比例尺:1μm。
H:aCTLA4/IL-15-Dex上IL-15和IL-15Rα的Western Blotting分析。
图3为aCTLA4/IL-15-Dex中(A)IL-15和(B)aCTLA4的定量分析(n=3)。
图4为用BMDC4T1-Dex,BMDCCT26-Dex,BMDCHCP-Dex,BMDCaCTLA4-Dex,BMDCIL-15-Dex和BMDCaCTLA4/IL-15-Dex刺激的脾脏T淋巴细胞的活化情况:
A:上清液中IFN-γ的定量。
B:上清液中IL-2的定量。
C:活化后的效应T细胞溶解4T1肿瘤细胞的效率,通过LDH释放细胞毒测定定量细胞溶解效率(n=4)。
D:活化后的效应T细胞溶解CT26肿瘤细胞的效率,通过LDH释放细胞毒测定定量细胞溶解效率(n=4)。
E:经各组外泌体孵育后用流式细胞术检测CFSE染色的活化T淋巴细胞的增殖情况。
F:经各组外泌体孵育后用流式细胞术检测CFSE染色的活化T淋巴细胞增殖的定量分析。
***P<0.001,**P<0.01,*P<0.05。
图5为流式细胞术检测各组外泌体孵育后CFSE染色的活化T淋巴细胞的增殖情况:
A:4T1-Dex,CT26-Dex和HCP-Dex孵育后T细胞增殖的流式分析结果。
B:4T1-Dex,CT26-Dex和HCP-Dex孵育后T细胞增殖的流式结果定量分析(**P<0.01,n=3)。
图6为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的体内外靶向能力的考察:
A:T淋巴细胞摄取Dex,IL-15-Dex,aCTLA4-Dex和aCTLA4/IL-15-Dex的共聚焦显微镜图像。
B,C:流式细胞术分析DiI标记的Dex,IL-15-Dex,aCTLA4-Dex和aCTLA4/IL-15-Dex的T淋巴细胞靶向效率。细胞核用Hoechst(蓝色)染色。比例尺:10μm。
图7(A)为静脉注射DiR标记的Dex,IL-15-Dex,aCTLA4-Dex和aCTLA4/IL-15后剖取各器官以及腋窝和腹股沟淋巴结的荧光成像和(B)荧光图像的ROI定量分析。
图8为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)抗肿瘤转移的治疗效果:
A:肺转移的体内生物发光图像。
B:各组治疗后剖取的肺脏的离体生物发光图像。
C:布式固定液对肺的染色图。
D:肺切片的H&E染色图。比例尺:100μm。
E:肝切片的H&E染色图。比例尺:100μm。
F:肺切片中转移面积百分比的定量分析(***P<0.001,**P<0.01,n=3)。
G:肝切片中转移面积百分比的定量分析(***P<0.001,**P<0.01,n=3)。
图9为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)抗肿瘤转移机制考察中流式细胞术分析血液CD4+Foxp3+Tregs细胞的比例变化。
A:流式细胞分析图片。
B:流式细胞分析的定量结果(***P<0.001,**P<0.01,n=4)。
图10为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)抗肿瘤转移机制考察中流式细胞术分析血液CD8+T细胞的比例变化。
A:流式细胞分析图片。
B:流式细胞分析的定量结果(**P<0.01,n=4)。
图11为偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)抗肿瘤转移机制考察中ELISA检测血浆IFN-γ水平(**P<0.01,n=4)。
图12为切除原发肿瘤后4T1-luc肿瘤的体内荧光成像。每组显示四只代表性小鼠。第10天的图像为手术前。
图13为(A)肿瘤生长曲线,(B)小鼠存活曲线和(C)体重变化曲线(***P<0.001,**P<0.01)。
图14为基于Luminex的血浆中细胞因子测定(n=4)。
图15为Saline和aCTLA4/IL-15-Dex组肿瘤浸润CD4+T细胞和CD8+T细胞的免疫荧光图像,比例尺:50μm。
图16为抑制肿瘤复发免疫机制的考察。流式细胞术分析肿瘤浸润的CD4+Foxp3+Tregs的比例变化(***P<0.001,**P<0.01,n=4)
A:流式细胞术分析图片。B:流式细胞术分析量化结果。
图17为抑制肿瘤复发免疫机制考察流式细胞术分析肿瘤浸润的CD8+T细胞的比例变化(**P<0.01,n=4)
A:流式细胞术分析图片。B:流式细胞术分析量化结果。
图18为抑制肿瘤复发免疫机制考察流式细胞术分析肿瘤浸润的CD8+CD62LlowCD44hi效应记忆T细胞的比例变化(**P<0.01,n=4)
A:流式细胞术分析图片。B:流式细胞术分析量化结果。
图19为4T1术后模型各组小鼠心脏,肝脏,脾脏,肺脏和肾脏的H&E染色图片。比例尺:100μm。
图20为(A)在CT26异位肿瘤模型治疗结束时解剖的CT26肿瘤照片。(B)肿瘤生长曲线,(C)肿瘤重量,(D)小鼠生存曲线(n=6)和(E)体重变化曲线。aCTLA4/IL-15-Dex组中第40天存活的小鼠再次接种CT26肿瘤细胞。将同周龄接种CT26肿瘤细胞的小鼠作为对照。(F)小鼠的肿瘤生长曲线(n=5)。结果显示为平均值±SD。(G)再次接种CT26肿瘤细胞的小鼠的生存曲线。***P<0.001,**P<0.01。
图21为流式细胞术分析CT26荷瘤小鼠肿瘤浸润的CD4+Foxp3+Tregs的比例变化(***P<0.001,n=5)。
A:流式细胞术分析图片。B:流式细胞术分析量化结果。
图22为流式细胞术分析CT26荷瘤小鼠肿瘤浸润的CD8+T细胞的比例变化(***P<0.001,**P<0.01,n=5)。
A:流式细胞术分析图片。B:流式细胞术分析量化结果。
图23为流式细胞术分析CT26荷瘤小鼠脾脏中CD8+CD62LlowCD44hi效应记忆T细胞的比例变化(***P<0.001,n=5)。
A:流式细胞术分析图片。B:流式细胞术分析量化结果。
图24为CT26肿瘤模型中Saline和aCTLA4/IL-15-Dex组肿瘤浸润的CD4+T细胞和CD8+T细胞的免疫荧光图像。比例尺:50μm。
图25为CT26肿瘤模型中治疗结束后各组小鼠心脏,肝脏,脾脏,肺脏和肾脏的H&E染色图片。比例尺:100μm。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将发明限制在所述的实施例范围之中。
实施例1:HSP70伴侣多肽(HCP)的提取和表征
采用免疫组化比较了正常组织和肿瘤组织中HSP70的表达。如图2A所示,HSP70在肿瘤组织中过表达,表明其伴侣多肽中存在丰富的肿瘤抗原。为了提取HCP,解剖分离肿瘤组织,并利用细胞筛网制备单细胞悬液。加入ACK裂解缓冲液去除红细胞。然后将制备的肿瘤细胞在含有蛋白酶抑制剂I的裂解缓冲液中冰上匀浆60分钟。在12000rpm下4℃离心30分钟后,使用抗HSP70抗体对上清液进行免疫沉淀并与A/G蛋白修饰琼脂糖珠孵育12小时。然后用裂解缓冲液洗涤蛋白质/抗体/琼脂糖珠复合物。将复合物与ATP溶液在25℃下孵育0.5小时。收集上清液,并用Bradford法进行蛋白质的定量测定。通过免疫沉淀技术在匀浆肿瘤组织中提取HCP,每克肿瘤组织可提取约50μg HCP。通过SDS-PAGE电泳对蛋白质进行分离,并使用考马斯蓝对凝胶染色。使用动态光散射检测分离出的HCP的粒径分布。如图2B-C所示,HCP的分子量约为50-70kDa,平均粒径约为10nm。
实施例2:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的制备及表征
从Balb/c骨髓中分离树突状细胞(DC)。冲洗小鼠股骨中的骨髓细胞,将细胞在含有10%胎牛血清,5ng/ml鼠IL-4和10ng/ml鼠GM-CSF的RPMI 1640培养基中培养,每2天半量换液。收集树突状细胞,并在第5天用无血清RPMI1640培养基铺在24孔板中,在37℃下用IL-15编码的重组腺病毒(rAD)和IL-15Rα编码的rAD(IL-15/IL-15RαrAD)共同转染细胞2小时。然后加入含10%胎牛血清,5ng/ml鼠IL-4和10ng/ml鼠GM-CSF的RPMI 1640培养基,并在37℃和5%二氧化碳的条件下继续培养至48小时。用含1μg/ml LPS的HCP刺激树突状细胞,然后在含有无外泌体胎牛血清的RPMI 1640培养基中进一步培养24h。使用超速离心提取工程化外泌体。具体地,将收集的细胞培养上清以330g离心12分钟去除细胞。将上清液以13000g离心25分钟去除细胞碎片。100000g超速离心110分钟后,收集工程化外泌体。分离的工程化外泌体用PBS洗涤并超速离心重悬。为了制备aCTLA4-生物素,将aCTLA4与sulfo-NHS-生物素在PBS中混合,并在室温下轻轻摇动反应0.5小时。使用超滤管纯化aCTLA4-生物素,在1600rcf下离心4分钟。将工程化外泌体分别与sulfo-NHS-生物素,链霉亲和素和aCTLA4-生物素反应1小时。并在PBS中洗涤3次,以去除每次反应之间的游离反应物(图1)。
蛋白质印迹法用于确认偶联免疫检查点阻断单克隆抗体基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的成功制备。使用SDS-PAGE电泳分离蛋白分子,并将蛋白质转移至PVDF膜上。用脱脂奶粉封闭PVDF膜并分别与抗TSG101,抗CD9,抗CD81,抗MHC I,抗MHCII,抗CD86,抗IL-15和抗IL-15Rα的一抗孵育。孵育二抗后,加入ECL化学发光试剂以检测免疫印迹。蛋白印迹分析表明,树突状细胞相关的蛋白(MHC I,MHC II和CD86)和外泌体相关的蛋白(TSG101,CD9和CD81)均在IL-15-Dex上表达,且腺病毒转染使外泌体上成功表达了IL-15和IL-15Rα(图2F、H)。使用ZetaView(德国Particle Metrix)进行了纳米颗粒跟踪分析(NTA)。纳米粒子追踪分析(NTA)显示IL-15-Dex的平均粒径为130nm,且粒径均一(图2D-E)。通过透射电子显微镜(TEM,Hitachi HT7700)观察了的形态。同时,用抗MHC II抗体进行了免疫胶体金标记。将样品与抗MHC II抗体在25℃下孵育2h,并在BSA溶液中洗涤3次。在25℃下与10nm金偶联的二抗孵育1小时后,用BSA洗涤3次。将样品用醋酸双氧铀染色并用TEM观察。为了证明aCTLA4和IL-15在aCTLA4/IL-15-Dex上的共定位,使用了绿色荧光蛋白(GFP)标记的IL-15/IL-15Rα编码的腺病毒转染树突状细胞,并提取外泌体。将aCTLA4修饰的外泌体与Alexa Fluor 594标记的二抗孵育。激光扫描共聚焦显微镜(CLSM)成像显示绿色和红色荧光的共定位(图2G),表明IL-15修饰的树突状细胞外泌体上成功偶联了aCTLA-4。使用BCA确定了aCTLA4/IL-15-Dex上修饰的aCTLA4的量,并用ELISA试剂盒检测了经0.01%SDS煮沸处理后aCTLA4/IL-15-Dex中IL-15的含量。在IL-15/IL-15Rα转染和aCTLA4修饰后,树突状细胞外泌体表面的IL-15和aCTLA4浓度显著升高。每100μg外泌体,可结合约1.8μg IL-15和1.4μg aCTLA4(图3A-B)。
实施例3:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)激活T细胞反应
将骨髓来源树突状细胞(BMDC)分别与4T1细胞裂解液刺激的树突状细胞外泌体(4T1-Dex),CT26细胞裂解液刺激的树突状细胞外泌体(CT26-Dex),HCP刺激的树突状细胞外泌体(HCP-Dex),HCP刺激的偶联aCTAL4抗体的树突状细胞外泌体(aCTLA4-Dex),HCP刺激的IL-15/IL-15Rα工程化树突状细胞外泌体(IL-15-Dex)和HCP刺激的偶联aCTAL4抗体的IL-15/IL-15Rα工程化树突状细胞外泌体(aCTLA4/IL-15-Dex)一起孵育。然后,将从Balb/c小鼠脾脏中收获的T淋巴细胞用上述BMDC处理3天。使用ELISA试剂盒检测上清液中IL-2和IFN-γ的量。使用LDH乳酸脱氢酶细胞毒性检测分析试剂盒评估活化的T淋巴细胞对4T1和CT26肿瘤细胞的细胞溶解效率。通过检测从裂解的肿瘤细胞中释放出的乳酸脱氢酶(LDH)来计算细胞毒性。结果如图4A-D所示,与4T1或CT26肿瘤细胞裂解物刺激的Dex孵育的BMDC(BMDC4T1-Dex或BMDCCT26-Dex)相比,HCP刺激的Dex孵育的BMDC(BMDCHCP-Dex)可以激活脾脏初始T细胞分泌更高水平的IL-2和IFN-γ。导致针对4T1和CT26肿瘤细胞的更高溶解效率。这些结果表明,提取的HCP是高度免疫原性的癌症抗原。此外,与HCP刺激的IL-15-Dex孵育的BMDC(BMDCIL-15-Dex)进一步增加了脾脏T细胞分泌的IL-2和IFN-γ,提高了肿瘤细胞溶解率。而与HCP刺激的aCTLA4/IL-15-Dex孵育的BMDC(BMDCaCTLA4/IL-15-Dex)激活的脾脏T细胞的IL-2和IFN-γ分泌比BMDCHCP-Dex增加了1.7倍,对4T1和CT26肿瘤细胞的溶解效率提高了2倍。表明aCTLA4/IL-15-Dex可通过树突状细胞间接激活初始T细胞。
考察了偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)直接刺激活化T淋巴细胞的能力。使用小鼠CD8+T细胞分离试剂盒提取aCTLA4/IL-15-Dex免疫后的Balb/c小鼠脾脏中的CD8+T细胞,并用CFSE染色。将4T1-Dex,CT26-Dex,HCP-Dex,aCTLA4-Dex,IL-15-Dex和aCTLA4/IL-15-Dex与CFSE标记的T细胞分别共培养72小时。流式细胞仪检测CFSE标记的T淋巴细胞的增殖情况。CFSE染色的流式细胞仪定量分析表明,HCP-Dex组的T细胞的增殖速率高于4T1-Dex和CT26-Dex组(图5A-B),确证了HCP的高免疫原性。与其他对照组相比,aCTLA4/IL-15-Dex实现了最有效的活化T细胞再刺激并增加了它们的增殖(图4E-F)。
实施例4:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)靶向T细胞和淋巴结
为了评估aCTLA4偶联后T细胞锚定的增强,用DiI将Dex,IL-15-Dex,aCTLA4-Dex和aCTLA4/IL-15-Dex染色。具体地,将Dex与5μg/ml DiI在37℃下孵育0.5小时。然后通过Exosome Spin Columns(MW 3000)除去游离染料。用DiI标记的Dex,IL-15-Dex,aCTLA4-Dex或aCTLA4/IL-15-Dex在4℃下与免疫后Balb/c小鼠脾脏中分离的CD8+T细胞孵育0.5h。将细胞用PBS洗涤3次,并重悬用于流式细胞仪分析。为了进行CLSM观察,将洗涤后的细胞转移至涂有聚L-赖氨酸的玻璃载玻片(Sigma-Aldrich)上,并用4%多聚甲醛溶液固定5分钟,用Hoechst 33342染核10分钟。PBS洗涤后,用CLSM观察细胞的荧光。aCTLA4-Dex和aCTLA4/IL-15-Dex均显示出比Dex和IL-15-Dex更高的DiI荧光强度,表明aCTLA4的修饰赋予外泌体增强的T细胞表面锚定能力(图6A)。使用流式细胞仪量化了T细胞靶向作用。如图6B-C所示,aCTLA4-Dex和aCTLA4/IL-15-Dex的荧光强度大约是Dex和IL-15-Dex的2倍,证实了aCTLA4修饰后外泌体增强的T细胞靶向能力。
实施例5:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)的体内分布
将Dex,IL-15-Dex,aCTLA4-Dex和aCTLA4/IL-15-Dex用DiR标记。具体地,将DiR与各组外泌体在37℃下孵育0.5小时,并使用Exosome Spin Columns(MW 3000)去除游离染料。将DiR标记的Dex,IL-15-Dex,aCTLA4-Dex和aCTLA4/IL-15-Dex静脉注射到免疫后的Balb/c小鼠体内。24小时后,收集腋窝和腹股沟淋巴结以及主要器官(心脏,肝脏,脾脏,肺脏和肾脏)进行离体荧光成像分析。从aCTLA4-Dex和aCTLA4/IL-15-Dex组中解剖的腋窝淋巴结和腹股沟淋巴结的荧光强度是Dex和IL-15-Dex组的2.5倍。同时,aCTLA4-Dex和aCTLA4/IL-15-Dex在给药后24小时脾脏的荧光强度是Dex和IL-15-Dex的1.9倍(图7A-B)。淋巴结和脾脏中蓄积的增强是由aCTLA4修饰赋予外泌体增强的T细胞锚定能力而实现的。
实施例6:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)抗肿瘤转移
将1×106个4T1-Luc乳腺癌细胞原位接种到雌性Balb/c小鼠的左侧乳腺脂肪垫中。荷瘤小鼠随机分为8组,在第7、10、13、16天以每只小鼠200μg Dex的剂量分别尾静脉注射生理盐水(Saline),aCTLA4,IL-15,Dex,aCTLA4/IL-15,aCTLA4-Dex,IL-15-Dex或aCTLA4/IL-15-Dex。为了模拟更恶性的侵袭和转移,在第10天将5×105个4T1-Luc细胞尾静脉注射到小鼠体内。在第20天,每只小鼠腹腔内注射3mg D-荧光素钾盐溶液,使用小动物活体成像系统检测原位肿瘤和肺转移情况。如图8A所示,Saline组小鼠在肺中显示出严重的转移。aCTLA4和IL-15组小鼠的肺和原发肿瘤部位的生物发光强度略有降低。Dex和aCTLA4/IL-15进一步提高了对转移的抑制作用。与Dex相比,aCTLA4-Dex和IL-15-Dex有效减轻了肿瘤的肺转移和原位生长。而aCTLA4/IL-15-Dex治疗消除了肺部的肿瘤转移信号。治疗结束后,在麻醉下处死小鼠并剖出新鲜的肺,在15mg/ml D-荧光素钾盐溶液中浸泡10分钟。通过生物发光成像检测肺部的肿瘤转移,aCTLA4/IL-15-Dex治疗的小鼠显示出微弱的生物发光信号(图8B)。同时,将肺在布氏固定液中染色,在肺脏离体布氏固定液染色中未观察到转移迹象,有效抑制了肿瘤的肺部转移(图8C)。对肺和肝切片进行H&E染色以评估抗转移效果,并使用ImageJ定量计算切片中的转移面积。如图8D-G所示,Saline组中的肿瘤细胞严重侵袭了肺脏和肝脏。aCTLA4和IL-15表现出中等的转移抑制能力。相比之下,Dex和aCTLA4/IL-15组的转移进一步减少。与Dex相比,aCTLA4-Dex和IL-15-Dex明显减少了转移性结节。aCTLA4/IL-15-Dex则最有效地抑制了肿瘤的转移,达到了协同治疗的效果。
为了验证体内T细胞的活化情况,使用小鼠外周血淋巴细胞分离试剂盒从血液中分离淋巴细胞。将细胞用荧光标记的抗CD3,CD4,Foxp3抗体染色以评估Treg含量的变化。将细胞与荧光标记的抗CD3,CD4,CD8抗体孵育检测CD8+T细胞。使用荧光标记的抗CD3,CD44,CD8,CD62L抗体对细胞染色分析记忆T细胞的变化。使用流式细胞仪检测染色的细胞,并使用FlowJo软件分析数据。同时使用ELISA试剂盒检测了血浆中IFN-γ水平。aCTLA4/IL-15-Dex有效减少了调节性T细胞(Treg),并显著增加了CD3+CD8+细胞毒性T淋巴细胞(CTL)的比例,提高了干扰素-γ(IFN-γ)的血浆水平(图9-11)。表明aCTLA4/IL-15-Dex可触发血液循环中T细胞介导的肿瘤抑制,从而避免全身性转移。
实施例7:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)抑制肿瘤术后复发
为了考察aCTLA4/IL-15-Dex抑制肿瘤术后复发的效果,将1×106个4T1-Luc肿瘤细胞接种到雌性Balb/c小鼠右侧乳腺脂肪垫中。在第10天,将小鼠随机分为8组,并以约1%的残留量切除肿瘤来模拟术后的微量残留。在第13、16、19、22天以每只小鼠200μg Dex的剂量分别尾静脉注射Saline,aCTLA4,IL-15,Dex,aCTLA4/IL-15,aCTLA4-Dex,IL-15-Dex和aCTLA4/IL-15-Dex。按照以下公式每两天用游标卡尺测定复发的肿瘤体积:1/2×长度×宽度2。在第10、11、20、30天,腹腔内注射D-荧光素钾盐溶液后10分钟,使用小动物活体成像观察肿瘤的生物发光。如图12、图13A-B所示,aCTLA4和IL-15无法有效抑制肿瘤术后复发。Dex和aCTLA4/IL-15组的生物发光强度有所降低。与Dex相比,aCTLA4-Dex和IL-15-Dex延缓了肿瘤的复发。一旦在手术后给予aCTLA4/IL-15-Dex治疗,手术部位的转移信号几乎消失了,同时也显著延长了小鼠的生存期。为了考察治疗的安全性,记录了治疗期间各组小鼠的体重,并剖取小鼠心脏,肝脏,脾脏,肺和肾脏进行H&E染色。aCTLA4/IL-15-Dex治疗小鼠的体重无明显变化,主要器官的H&E染色未发现明显异常(图13C、图19)。在第32天,将Saline和aCTLA4/IL-15-Dex组小鼠的残留肿瘤切除并速冻。然后,用冷冻切片机切开肿瘤组织,将其固定在载玻片上,在4℃下使用CD4和CD8一抗孵育过夜,加入荧光标记的二抗后,通过CLSM观察样品。免疫荧光染色显示aCTLA4/IL-15-Dex治疗后残存肿瘤组织的CD4+和CD8+T细胞浸润明显增多(图15)。收集外周血并以480g离心12分钟。取上清液,通过Luminex测定了各种细胞因子的水平。IFN-γ,TNF-α,IL-1β,IL-2,IL-6,IL-12P70,IL-18和GM-CSF的定量表明血浆中细胞因子的水平显著升高,进一步证实了aCTLA4/IL-15-Dex可以有效促进适应性免疫应答(图14)。使用小鼠肿瘤浸润淋巴细胞分离试剂盒从肿瘤中分离淋巴细胞,检测肿瘤浸润的Treg,CD8+T细胞,记忆T细胞。与其他对照组相比,aCTLA4/IL-15-Dex治疗进一步上调了CD8+T细胞的百分比,而CD4+Foxp3+Treg的百分比则显著降低(图16-17)。此外,我们检测了治疗后CD8+效应记忆T细胞(TEM,CD8+CD62LlowCD44hi)的变化。如图18所示,aCTLA4/IL-15-Dex治疗显著增加了次级淋巴组织(脾脏)中的TEM细胞水平,表明产生了持久的保护性免疫记忆。
实施例8:偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体(aCTLA4/IL-15-Dex)产生持久的抗肿瘤免疫记忆
在Balb/c小鼠皮下接种1×106个CT26肿瘤细胞,在肿瘤体积达到约100mm3后,将小鼠随机分为8组。在第0、3、6、9、12天以每只小鼠200μg Dex的剂量分别尾静脉注射Saline,aCTLA4,IL-15,Dex,aCTLA4/IL-15,aCTLA4-Dex,IL-15-Dex和aCTLA4/IL-15-Dex。每两天测量一次肿瘤体积,每天观察小鼠存活率。在第20天处死小鼠,剖出肿瘤并称重。如图20A-C所示,Saline组肿瘤体积明显增加,在第20天为约1460mm3。aCTLA4和IL-15组的肿瘤大小分别达到约1190mm3和1120mm3,对肿瘤生长的抑制作用较差。在治疗结束时,Dex和aCTLA4/IL-15进一步减慢了肿瘤的生长,肿瘤平均体积分别为约820mm3和850mm3。与Dex相比,aCTLA4-Dex和IL-15-Dex显著增强了对肿瘤的抑制作用,肿瘤体积约为530mm3和480mm3。此外,aCTLA4/IL-15-Dex表现出最佳的肿瘤抑制效果,第20天肿瘤体积约为215mm3,且在40天内显示出较高的存活率(图20D)。记录治疗期间小鼠体重并在治疗结束后剖取各组小鼠主要器官(心脏,肝脏,脾脏,肺脏,肾脏)进行H&E染色。小鼠体重无异常波动且H&E染色的切片上未观察到明显的病变,表明aCTLA4/IL-15-Dex的安全性良好(图20E、图25)。接着考察了不同组小鼠之间TILs的差异。如图22所示,与Saline相比,aCTLA4和IL-15轻微增加了肿瘤浸润CD8+T细胞的水平。而在Dex和aCTLA4/IL-15组中检测到升高的肿瘤浸润CD8+T细胞水平。与Dex相比,aCTLA4-Dex和IL-15-Dex进一步促进了效应T细胞的产生,从而产生了更强的抗肿瘤活性。aCTLA4/IL-15-Dex组小鼠CD8+T细胞的百分比最高,与其最佳的抗肿瘤效果一致。免疫荧光染色也证明了aCTLA4/IL-15-Dex组小鼠的肿瘤浸润CD4+和CD8+T细胞的显著增加(图24)。同时,aCTLA4/IL-15-Dex治疗后免疫抑制性Treg的水平持续降低(图21)。通过流式细胞仪检测了各组治疗后脾脏中TEM细胞的水平。如图23所示,aCTLA4/IL-15-Dex治疗可刺激最多的TEM细胞,有效的TEM诱导有助于长期抗肿瘤免疫记忆的产生。
为了证明aCTLA4/IL-15-Dex介导持久的抗肿瘤免疫记忆,在aCTLA4/IL-15-Dex组中40天后仍存活的小鼠皮下接种1×106个CT26肿瘤细胞。作为对照,给同龄小鼠皮下接种CT26肿瘤细胞。记录小鼠的肿瘤体积和存活率。如图20F-G所示,aCTLA4/IL-15-Dex组中幸存的小鼠明显抑制了重新接种的肿瘤的生长,并且存活时间显著延长。这说明aCTLA4/IL-15-Dex治疗可诱导持久的抗肿瘤免疫记忆。
Claims (10)
1.偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体,其特征在于,所述的肿瘤治疗外泌体是以肿瘤抗原激活、基因编码的重组腺病毒转染的树突状细胞外泌体为载体,以生物素-链霉亲和素-生物素复合物为连接臂,将aCTLA4抗体修饰到外泌体表面而得的aCTLA4修饰的肿瘤治疗外泌体。
2.如权利要求1所述的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体,其特征在于,所述的树突状细胞为骨髓来源的树突状细胞、外周血树突状细胞、DC2.4中的至少一种;所述的肿瘤抗原为HSP70伴侣多肽、OVA、MAGE以及通过细胞或组织破碎提取出来的肿瘤特异性抗原中的至少一种;所述的基因为IL-15、IL-15Rα中的至少一种。
3.如权利要求1所述的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体,其特征在于,树突状细胞外泌体、IL-15、aCTLA4的质量比为1-2:1-2:80-130,优选为1.5-1.8:1.4-1.6:100-110。
4.如权利要求1所述的肿瘤治疗外泌体的制备方法,包括以下步骤:
(1)培养树突状细胞,将骨髓细胞均匀分散在细胞培养板中培养,使用GM-CSF和IL-4诱导骨髓细胞分化为树突状细胞;
(2)提取外泌体:用基因编码的重组腺病毒转染树突状细胞,并用抗原刺激树突状细胞成熟,收集细胞培养上清,经过超速离心法提取外泌体;
(3)aCTLA4抗体修饰外泌体:将外泌体分别与sulfo-NHS-生物素,链霉亲和素和aCTLA4-生物素反应1-2小时,并在PBS中洗涤三次,以除去每次反应间的游离物;所述的aCTLA4-生物素由aCTLA4与sulfo-NHS-生物素在PBS中反应得到。
5.如权利要求4所述的制备方法,其特征在于,步骤(1)中,在细胞培养板中加入含有10%胎牛血清、10-30ng/ml的GM-CSF和10-30ng/ml的IL-4的RPMI1640培养基,每隔两天用含有20-60ng/ml的GM-CSF和20-60ng/ml的IL-4的新鲜培养基半量换液。
6.如权利要求4所述的制备方法,其特征在于,步骤(2)中,用基因编码的重组腺病毒转染树突状细胞48小时后,用含有0.2-0.6mg/ml的肿瘤抗原与树突状细胞孵育12-24小时后,更换含有无外泌体血清的细胞培养液继续培养24-48小时并收集上清液提取外泌体。
7.如权利要求4所述的制备方法,其特征在于,步骤(3)中,利用10kDa超滤离心管将aCTLA4-生物素溶液离心除去游离的生物素。
8.权利要求1-3任何一项所述的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体在制备药物传递系统中的应用。
9.权利要求1-3任何一项所述的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体在制备抗肿瘤或抗肿瘤转移药物中的应用。
10.权利要求1-3任何一项所述的偶联免疫检查点阻断抗体的基因工程化肿瘤治疗外泌体在制备注射给药、口服给药或局部给药系统中的应用。
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