CN116920082A - 基于酵母细胞外壳的新抗原肿瘤治疗性疫苗 - Google Patents
基于酵母细胞外壳的新抗原肿瘤治疗性疫苗 Download PDFInfo
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Abstract
我们通过GP上羟基与含环氧基PEG1的开环反应将N3偶联到GP外壳,得到GP‑N3;然后将含有半胱氨酸的多肽与含有DBCO和马来酰亚胺的PEG2连接,得到DBCO‑多肽;最后将二者通过点击化学连接,得到了一种新型的基于酵母外壳的新抗原多肽疫苗体系GP‑peptide。该体系制备简单且批次稳定、尺寸均一,可以被抗原呈递细胞高特异性摄取。通过与TLR激动剂的联合,该体系在动物模型上显示出强大的CTL免疫激活能力,并在小鼠淋巴瘤、黑色素瘤、乳腺癌及结肠癌模型上有效抑制肿瘤生长,甚至完全清除肿瘤,诱导长期的肿瘤排斥记忆。这些结果为其进一步临床推广和个性化疫苗治疗提供了广阔的可能性。
Description
所属技术领域:
本发明涉及肿瘤学、免疫学相关技术领域,具体涉及基于酵母细胞外壳的新抗原肿瘤治疗性疫苗的制备及其临床前小鼠水平评估。
背景技术:
相比于正常组织中也会表达的肿瘤相关抗原,新抗原(peptides)被认为是诱导肿瘤免疫排斥反应的理想抗原。它是一类由肿瘤基因非同义点突变产生的与人类白细胞抗原结合的肽,是癌症特异性抗原,它们不受中枢免疫耐受的影响,具有高度的免疫原性;它们不存在于正常组织中,所以能够摆脱中枢胸腺免疫耐受的影响,同时也可以避免自身免疫性疾病的风险并能产生强烈的抗肿瘤免疫应答。因此,近些年来针对新抗原的策略是使癌症治疗性疫苗获得重要突破,如今基于肿瘤新抗原的个性化肿瘤疫苗(PCV)已成为当前癌症免疫治疗的一个重要分支,并有大量的临床实验正在开展,取得了一系列令人振奋的结果。
Ugur和Catherine J.Wu等两个团队同时将新抗原多表位疫苗首先推上了针对“热肿瘤”的临床实验。Ugur团队制备了mRNA新抗原多表位疫苗,并对2例晚期黑色素瘤患者进行了疫苗接种后,结果显示疫苗诱导了良好的T细胞浸润和对自体肿瘤细胞的新表位特异性杀伤。转移事件的累积发生率大大降低,并且产生了持续的无进展生存。五名转移性疾病患者中有两名产生与疫苗相关的客观反应,另外一名患者在接种疫苗和PD-1单抗抑制剂后出现完全缓解。Catherine J.Wu团队合成了基于13-20个新抗原长肽的治疗性疫苗,并用TLR3 激动剂Poly-I:CLC作为免疫佐剂,对晚期黑色素瘤病人进行了疫苗接种。在6名接种疫苗的患者中,4名在接种后25个月没有复发,而2名复发的患者随后接受了抗PD-1单抗注射治疗,并经历了肿瘤完全消退,新抗原特异性T细胞库扩大。一年后Ugur和Wu的团队又分别进一步报道了他们在“冷肿瘤”中的临床试验结果。与黑色素瘤治疗方案相似,美国Catherine J.Wu团队合成了基于多个个新抗原的长肽疫苗,并采用Poly-I:CLC作为佐剂,并对手术切除和常规放射治疗后新诊断为I/Ib期胶质母细胞瘤的患者进行免疫接种。结果显示该疫苗方案能产生循环的多功能的新抗原特异性CD4和CD8T细胞反应,这些反应丰富了记忆表型,并显示出肿瘤浸润性T细胞数量的增加。利用单细胞T细胞受体分析,他们的结果还显示来自外周血的新抗原特异性T细胞可以迁移并靶向颅内的胶质母细胞瘤的证据。但非常不理想的是,该疫苗的接种对于恶性胶质瘤的疗效并不显著。因为新抗原疫苗制备流程较为繁琐,生产周期长,因此晚期病人可能等不了这么久,针对这个问题,Ugur Sahin团队率先采用了新策略进行临床试验:他们在个性化新抗原疫苗生产的过程中,率先使用生产好的“现货型”疫苗对胶质瘤病人先进行一轮接种,然后在新抗原疫苗生产出来以后,再用得到的新抗原疫苗进行之后几轮疫苗接种。之后他们再病人外周血中检测到了肿瘤新抗原特异性T细胞,并且观察到这些病人的生存期明显延长。除此以外,还有非常多的临床试验正在如火如荼的进展之中。但较低的抗原特异性细胞毒性T淋巴细胞(CTL)的免疫应答效率极大限制了其临床应用。其主要原因是多肽抗原的免疫原性弱,半衰期短,所以很难在体内诱导足够强的免疫反应。因此,如何提高机体产生肿瘤特异性CTL的数量和质量是当前提高癌症治疗性疫苗应用潜力的一个技术瓶颈。
为了解决这一关键问题,我们提出了一种基于酿酒酵母来源的β-(1,3)-葡聚糖颗粒 (Glucan particles,GPs)的肿瘤新抗原疫苗递送系统。GP来自于天然的食用酿酒酵母,其主要成分β-葡聚糖是一种高度安全的物质,被美国食药监管局(FDA)批准成为了公认安全 (GRAS)的可添加食品,广泛存在于真菌、酵母、植物和细菌的细胞壁中,也存在于燕麦和大麦等谷物中。在哺乳动物中,它们被中性粒细胞、巨噬细胞和树突状细胞上的免疫细胞受体如Toll样受体、Dectin-1、CR3和CD5识别为病原体相关的分子模式,或被清道夫受体CD5、CD36和SCARF1等识别。β-(1,3)和β-(1,6)连接的葡聚糖都能有效地激活补体的替代途径,导致真菌调理和趋化因子的产生,从而招募炎症细胞,是已知的天然免疫系统的有效激活剂。例如,在小鼠模型中,连续2周每天口服0.1mg/kg的来自面包酵母的β-葡聚糖微颗粒显著增加腹膜巨噬细胞的吞噬活性。基于此,GPs有可能是一种非常有潜力的负载新抗原的疫苗载体。
发明内容:
我们发明了一种新型的基于酵母多肽外壳颗粒(GP)的新抗原多肽的疫苗载体体系GP- peptide。制备过程:我们先将酿酒酵母溶于ddH2O中,用离心/重悬的方法清洗三遍除去酵母细胞外的化学物质,然后通过氢氧化钠-盐酸的先后碱酸处理将酵母内部原有的内含物质全部溶解去除,得到中空、均一的多糖颗粒(Glucan particles,GPs)。然后,为了将新抗原模式多肽OVA257-264通过共价化学偶联的方式装载到GP的多糖颗粒上,我们成功的通过 GPs上丰富的羟基(OH)与PEG1分子上的环氧基(Epoxy)在碱性条件下的环氧开环反应将叠氮官能团(N3)偶联到GP多糖外壳上,得到GP-N3。同时,OVA257-264多肽通过在N 末端引入半胱氨酸(Cysteine)来与两端分别含有马来酰亚胺活性接头和DBCO接头的分子 PEG2分子反应,得到末端修饰有DBCO的peptide(DBCO-peptide)。最后,我们将得到的GP-N3和DBCO-peptide混合均匀,通过叠氮和DBCO上三键之间的点击化学反应在室温下反应2小时就能实现新抗原多肽与GPs的高效偶联,得到GP-peptide多肽疫苗颗粒。对于 MHCI限制性的OVA257-264模式多肽、MHCII限制性的OVA323-339模式多肽以及B16F10黑色素瘤新抗原M30、4T1乳腺癌新抗原M25和CT26结肠癌新抗原ME1和ME4,我们均通过以上方式与GP高效偶联,得到GP-peptide疫苗颗粒体系。
GP-peptide可以激发机体产生强效的抗原特异性CTL细胞免疫反应,并有效的用于肿瘤的治疗。与传统的基于各种合成的纳米颗粒的疫苗相比,该颗粒体系制备相对简单而且批次之间稳定,颗粒大小均一,并具有树突状细胞和巨噬细胞等抗原呈递细胞摄取的高度特异性。通过与TLR受体激动剂PolyI:C或CpG 2395的联合,尤其是后者,该颗粒疫苗体系在动物模型上显示出强大的免疫激活能力,高效率激发机体产生针对各类新抗原的特异性CTL 免疫应答,并在EG7·OVA淋巴瘤模型和B16F10黑色素瘤,4T1乳腺癌及CT26结肠癌等多种小鼠同系肿瘤模型上,高效率的抑制肿瘤的生长。值得一提的是,在该策略可以在多个小鼠模型上实现肿瘤的完全清除,并在获得肿瘤除的小鼠体内诱导了长期的肿瘤排斥记忆的能力,完全避免再次接种肿瘤的生长。这些结果为其进一步临床推广和个性化疫苗治疗提供了广阔的可能性。
附图说明
图1是GP-peptide疫苗颗粒的制备过程分子原理及所用原料分子式;
图2是通过HPLC监测GP-OVA257-264制备过程中的反应效率。(A)Cysteine-OVA257-264与PEG2的反应效率监测。(B)DBCO-OVA257-264与GP-N3的反应效率监测,下面的线代表二者反应后的上清液,箭头所指代表实际的DBCO-OVA257-264峰,其他的小峰是上一步反应中过量的原料峰;
图3是GP-OVA257-264制备过程中(GP、GP-N3和GP-OVA257-264)的扫描电子显微镜图像和透射电子显微镜图像;
图4是用GP-OVA257-264-Cy5和OVA257-264-Cy5腹股沟免疫小鼠后24、48、72小时后小鼠的引流淋巴结活体成像图;
图5是流式细胞仪检测GP-OVA257-264诱导BMDCs表达MHCI、MHCII、CD40、 CD80、CD86等表面活化分子;
图6是流式细胞仪分析GP-OVA257-264对OT-1CD8T细胞的增殖,OT-1体外增殖实验的示意图(A)和GP-OVA257-264及其他对照组疫苗在体外对OT-1小鼠CD8T细胞的增殖流式图(B),OT-1体内增殖实验的示意图(C)和GP-OVA257-264及其他对照组疫苗在体内对OT- 1小鼠CD8T细胞的增殖流式图(D);
图7是流式细胞仪分析GP-OVA257-264对C57BL/6野生型小鼠的抗原特异性CD8T细胞的增殖、活化的促进效果,OVA257-264抗原特异性CD8T细胞的流式散点图(A)及数量统计图(B)。流式分析免疫后初始CD8T细胞(C)和中央记忆T细胞(D)的数量变化;
图8是免疫后小鼠全脾细胞体外用OVA257-264多肽抗原再刺激后的Elispot检测;
图9是ELISA监测C57BL/6小鼠被GP-OVA323-339、OVA323-339、OVA全长蛋白免疫不同次数后总IgG(A-B)、IgG1(C-D)、IgG2a(E-F)的OVA特异性抗体滴度;
图10是GP-OVA257-264的抗肿瘤效果检测,EG7·OVA预防性淋巴瘤模型的建立示意图 (A),肿瘤抑制效果(B)和小鼠生存率(C);以及GP-OVA257-264与不同佐剂联合后的肿瘤抑制效果(D)和小鼠生存率(E);
图11是GP-M30的抗肿瘤效果检测,B16F10黑色素瘤转移瘤模型的建立示意图(A),GP-M30与PolyI:C、CpG 2395的联合免疫后的小鼠肺部图像(B)和肺转移灶个数(C);
图12是GP-M25的抗肿瘤效果检测,4T1乳腺癌皮下肿瘤模型的建立示意图(A),GP-M25与CpG 2395的联合免疫后的小鼠肿瘤生长曲线(B)和肿瘤浸润T细胞分析(C);
图13是GP-ME1-ME4的抗肿瘤效果检测。CT26结肠癌皮下肿瘤模型的建立示意图(A),GP-ME1-ME4与CpG 2395的联合免疫后的整体肿瘤生长曲线(B),生存率和肿瘤完全抑制率(C)以及不同疫苗免疫组中单只小鼠的肿瘤生长曲线(D),箭头表示疫苗接种时间点。
具体实施方式
本领域技术人员会理解,下述实施例用于进一步说明本发明但不意味着限制本发明。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器均为可以通过市场获得的常规产品。
缩写语
peptide:新抗原;rpm:每分钟转速;N3:叠氮基;PEG:聚乙二醇;Epoxy:环氧基团;Maleimide:马来酰亚胺;DMSO:二甲基亚砜;HPLC:高效液相色谱;GM-CSF:粒细胞巨噬细胞集落刺激因子;IL:白介素;IFN-γ:干扰素-γ;BMDC:骨髓来源的树突状细胞; MHC:主要组织相容性复合体;CD:白细胞分化抗原;APC:抗原递呈细胞;Elispot:酶联免疫斑点实验;tetramer:多肽-MHCI复合四聚体;HRP:辣根过氧化物酶;TMB:四甲基联苯胺;PolyI:C:聚肌苷酸-聚胞苷酸;CpG:胞嘧啶鸟嘌呤二核苷酸;TLR:toll样受体。
下面用实施例进一步描述本发明,但所述实施例仅用于本发明而不是限制本发明。
实施例1
简称为β-葡聚糖颗粒GP-新抗原颗粒(GP-peptide)的制备(附图1)
(1)酵母来源的β-葡聚糖颗粒外壳(GP)的制备
将40克酿酒酵母S.cerevisiae重悬于双蒸馏水(ddH2O)中,用ddH2O洗涤3次,每次2000rpm离心5分钟。再将沉淀重悬于1M NaOH溶液中,在80℃下搅拌1小时。用ddH2O洗涤沉淀3次,每次2000rpm离心5分钟。用1M盐酸调节pH值至4-5,在55℃下搅拌1小时,3500rpm离心5分钟。后用ddH2O洗涤3次,3500rpm离心5分钟,异丙醇再悬浮5次,异丙醇洗涤5次,3500rpm离心2分钟。沉淀重悬在丙酮中,用丙酮洗涤两次,每次3500rpm离心2分钟。最后,将得到的葡聚糖颗粒(GPs)在通风柜中自然干燥,分散在研钵中,室温下干燥保存。
(2)GP-N3的制备
将葡聚糖颗粒(GPs)重新悬浮于0.5M氢氧化钠溶液中,室温搅拌30分钟,然后加入等量的N3-PEG4-Epoxy Linker,室温持续搅拌一夜。5000rpm离心3分钟,弃去上清。将沉淀再悬浮于ddH2O中,用盐酸调整pH至2.5,停止反应。然后用ddH2O洗涤三次沉淀,再用DMSO洗涤三次,离心再悬浮。最后,将微球重新悬浮在DMSO中,得到25mg/mL GP- N3溶液。
(3)新抗原多肽的DBCO修饰
首先,通过体外合成C端含有半胱氨酸残基的新抗原多肽:OVA257-264、OVA323-339、4T1-M25、CT26-ME1、CT26-ME4、B16-M30,然后将新抗原多肽在DMSO中溶解至终浓度为10mM的溶液。DBCO-PEG4-Maleimide马来酰亚胺也溶解在DMSO中至10mM溶液中。将半胱氨酸-多肽与DBCO-PEG4-maleimide等量比1.1∶1共同加入,再加入0.5%(v/v) 的N,N-二异丙基乙胺(DIPEA)。25℃静置2小时,然后采用高效液相色谱(HPLC)测定反应效率。反应完成后,将得到的DBCO-peptide用乙醚沉淀,自然干燥,然后将沉淀再溶解在DMSO溶液中。
(4)GP-N3和DBCO-peptide分子的偶联反应及监测
将GP-N3和DBCO-peptide根据我们需要的装载剂量简单混合后,加入小磁子,置于磁力搅拌器中搅拌2小时,即得到GP-peptide疫苗颗粒。然后,我们先后用无菌的ddH2O和无菌磷酸盐缓冲液(Phosphate buffer saline,PBS)用重悬/离心的方法将偶联反应完全的GP- peptide颗粒洗涤5次,共十次,以将DMSO从反应溶液中完全除掉。最后,我们将得到的GP-peptide颗粒用无菌PBS或无菌生理盐水重悬,并仅有用于细胞孵育或小鼠免疫。
实施例2
简称为GP-N3和DBCO-peptide分子的偶联反应效率的高效液相色谱法监测(附图2)
在GP-N3和DBCO-新抗原分子的偶联反应后,8000rpm,离心5分钟,用高效液相色谱法 (High Performance Liquid Chromatography,HPLC)测定上清液中剩余的DBCO-peptide,以观察反应效率。
实验结果及结论:两者对比可知,实际的DBCO-peptide峰在反应后几乎完全消失,而其余的小杂质峰的位置、高度跟反应前相比几乎没有任何变化。这直接证明了DBCO-peptide与 GP-N3的点击化学反应确实成功了,而且迅速高效。
实施例3
简称为GP-peptide的电子显微镜表征(附图3)
将GP、GP-N3和GP-OVA257-264悬浮于ddH2O中,超声处理30分钟,使颗粒分散均匀。将所得溶液用ddH2O稀释到适当浓度,滴加到透射电子显微镜专用铜网上的碳支撑膜上,置于冷冻干燥机中冻干,然后用扫描电子显微镜观察。
将GP、GP-N3和GP-OVA257-264悬浮于ddH2O中,超声处理30分钟,使颗粒分散均匀。然后3000rpm离心5分钟,除去上清,之后依次在30%、50%、70%、80%、90%、 100%乙醇溶液中浸泡3次,每次浸泡15分钟,浸泡完成后通过3000rpm离心5分钟,除去上清,使其完全脱水。然后将脱水的GP、GP-N3和GP-OVA257-264用乙醇稀释至适当浓度,滴于载玻片上,自然干燥,在20千伏电压下的透射电子显微镜下观察。
实验结果及结论:在修饰了叠氮基团和OVA257-264新抗原多肽后,GP颗粒的内部构造和颗粒尺寸几乎未发生变化,但是其通透度明显降低,不能非常清晰的观察到其内部构造,这进一步间接证实了叠氮基团和OVA257-264新抗原多肽与GP颗粒的成功偶联
实施例4
简称为GP-peptide疫苗在小鼠体内的淋巴结迁移检测(附图4)
将Balb/c小鼠随机分组,在腹股沟皮下接种GP-OVA257-264-Cy5或OVA257-264-Cy5进行活体成像定位。使用动物活体成像系统监测24小时、48小时或72小时注射部位和引流淋巴结的荧光信号。
实验结果及结论:在注射部位的引流淋巴结中,随时间的延长,GP-OVA257-264-Cy5注射组小鼠的荧光信号持续增强,而其他器官中几乎没有显著的荧光信号;而游离的OVA257-264-Cy5 注射组小鼠引流淋巴结中几乎没有Cy5荧光出现。
实施例5
简称为GP-peptide在体外对抗原递呈细胞的活化效果检测(附图5)
首先我们在无菌条件下取出C57BL/6小鼠的股骨、股骨和腓骨。然后将其转移到细胞培养皿中,并用一次性无菌注射器从股骨、股骨和腓骨中取出骨髓,用移液枪将其吹打均匀,分散成单细胞悬液,置于含有2%青霉素和链霉素的1640培养基中。然后将细胞在水平转子离心机中1400rpm离心7分钟,弃上清,后用2mL红血球裂解液重悬细胞,静置5分钟,之后加入8mL无菌PBS停止裂解,然后继续1400rpm离心7分钟,弃上清。接下来我们将收集的原代骨髓细胞重新悬浮于1640完全培养液中,培养液中含有5%(v/v)热灭活胎牛血清、1%(v/v)青霉素和链霉素,40ng/mL GM-CSF,20ng/mL IL-4和50μM β-巯基乙醇,在37℃、5%CO2浓度的无菌细胞恒温培养箱中培养6天。每隔两天,要小心翼翼地用新鲜的东西替换一半熟的。在整个文化培养过程中,应该避免摇晃。第6天,收集所有悬浮细胞和半贴壁细胞并计数,以备后续实验使用。
将培养成熟的BMDCs培养在10厘米直径的24孔细胞培养皿中,接种密度为1×106细胞/mL,每孔加入500μL。12小时后分别加入PBS、OVA257-264、GP和GP-OVA257-264,孵育 24小时。1400rpm离心5分钟,取上清液,用酶联免疫吸附(ELISA)测定试剂盒检测IL-6 和IL-12p70的表达。同时我们收集细胞,PBS洗涤2次,然后分别用FITC conjugated anti- mouseCD11c抗体标记BMDC细胞,同时联合荧光标记抗小鼠CD80、CD86、CD40、H- 2Kb/H-2Db(MHCI)、I-A/I-E(MHCII)对BMDC和BMDM的表面活化分子进行染色,然后用流式细胞分析技术分析细胞。
实验结果及结论:BMDCs细胞表面的MHCI、MHCII、CD80、CD86和CD40等共刺激信号的表达都显著上调;GP-OVA257-264颗粒可以显著促进APC细胞的免疫激活。
实施例6
简称为GP-peptide在体内外对OT-1转基因CD8T细胞的增殖检测(附图6)
在分选OT-1细胞之前24小时,我们先将疫苗颗粒及其对照材料与分化成熟后的BMDC细胞在24孔板中共培养24小时,以提供抗原负载的DC供实验用。孵育过夜后,我们取OT-1TCR转基因老鼠,将其安乐死,无菌条件下(酒精消毒后浸泡于含酒精的培养皿中)取其脾脏并制备全脾细胞悬液。然后我们使用小鼠CD8T细胞分离试剂盒对得到的小鼠全脾细胞进行分选,得到OT-1小鼠的脾脏CD8T细胞(OT-1细胞)。分离的OT-1细胞用5 μM羧基荧光素琥珀酸酯(CFSE)进行染色,简单来说,将分离后的OT-1细胞重新悬浮在 5μM CFSE的PBS溶液中,使得细胞密度为2×106细胞/mL。然后将其用锡箔纸包裹避光,在37℃下孵育15分钟。15分钟后加入等量的预冷的PBS缓冲液,然后1400rpm,离心7 分钟,之后再用PBS溶液洗涤细胞两遍离心转速时间均不变,得到CFSE标记的OT-1细胞。最后将与疫苗孵育后的BMDCs与CFSE染色的OT-1细胞以1∶10的比例共培养于含 10%(v/v)胎牛血清、1%(v/v)青霉素和链霉素、50μM β-巯基乙醇的RPMI 1640细胞中,96孔圆底板共培养72小时。然后用用流式细胞分析仪检测测定CFSE荧光的稀释程度来T细胞增殖效率,稀释比例越高,证明其增殖比例越高。
为了在小鼠体内研究小鼠免疫后的OT-1细胞的增殖,我们首先如(1)中所述分离OT- 1转基因小鼠脾细胞然后用5μM CFSE标记。然后我们取C57BL/6野生型小鼠,并将CFSE标记的OT-1细胞静脉回输到免疫后的C57BL/6小鼠(每只小鼠1×107细胞)体内。在回输后24小时,我们对小鼠随机分组并在腹股沟皮下接种,在接种后72小时,对受体小鼠实施安乐死,用流式细胞仪检测测定CFSE稀释程度以检测T细胞增殖效率。
实验结果及结论:GP-OVA257-264在体外可诱导50%以上的CD8 T细胞增殖率,而GP仅能诱导7%左右的细胞增殖率;GP-OVA257-264在体内可诱导32.4%的CD8 T细胞增殖率,而GP 仅能诱导0.7%的CD8 T细胞的增殖
实施例7
简称为GP-peptide在体内诱导小鼠细胞免疫应答的检测(附图7)
将C57BL/6小鼠(对于新抗原OVA257-264或M30)和Balb/c小鼠(对于新抗原M25或ME1、ME4)随机分组,每14天在腹股沟皮下注射两次GP-peptide颗粒及对应的对照疫苗分子。第二次接种后7天,处死小鼠,取其全脾细胞检测细胞免疫反应。我们用PE conjugatedanti-mouse CD8、FITC conjugated anti-mouse CD62L和AF647 conjugated anti- mouseCD44流式抗体对小鼠脾细胞进行染色后用流式细胞仪分析记忆T细胞的分化情况, CD62L+CD44-代表初始T细胞,CD62L+CD44+代表中央记忆T细胞(TCM), CD62L-CD44+代表效应记忆T细胞(TEM)。我们用PE conjugated H-2Kb-OVA257-264四聚体染色30分钟,然后加入小鼠FcR封闭抗体(Anti-mouse CD16/CD32 antibody),4℃孵育5 分钟,再加入FITCconjugated anti-mouse CD8抗体(注意:此步CD8抗体不可用Clone 53- 6.7克隆号的抗体染色,因为这个克隆的抗体会导致假阳性结果的出现。可以选择Clone KT15,其与H-2Kb-OVA257-264tetramer的共同染色效果较好),4℃染色20分钟,PBS洗涤2 次,用流式细胞仪分析CD8T细胞中的OVAtetramer阳性细胞比例的变化。
实验结果及结论:在GP-OVA257-264免疫组中,CD8 T细胞中MHCI-OVA257-264四聚体阳性细胞的比例显著上升,是OVA257-264免疫组和其他对照组中的比例的5倍以上.这证明相比OVA257-264,GP-OVA257-264能够显著诱导小鼠体内OVA257-264特异性的CD8 T细胞产生。同时在GP-OVA257-264免疫组中,初始的CD8 T细胞比例明显降低,而这部分降低的初始CD8 T 细胞则可以更显著的被诱导分化为中央记忆性CD8 T细胞,意味着抗原特异性细胞免疫质量的提升和免疫记忆的增强
实施例8
简称为GP-peptide在体内诱导小鼠IFN-γ特异性Elispot应答的检测(附图8)
为了监测小鼠体内OVA或肿瘤新抗原特异性细胞免疫应答,我们先将MultiScreen-IP过滤板提前一天用35%乙醇活化30秒,然后每孔包被1.5μg抗体AN18过夜。次日,将5×105小鼠脾细胞加入孔中,加入10μM新抗原肽或2×104肿瘤细胞,37℃共孵育48小时。孵育后,将孔中的细胞取出。PBS洗板5次,每孔加入1.0μg/mL生物素化检测抗体R4-6A2 100 μL,室温孵育2小时,PBS洗5次。每孔加入100μL链霉亲和素偶联的HRP,室温孵育1小时后,再加入每孔100μL TMB单组分显色液显色15-30分钟,待有明显斑点出现时,将显色液倒掉,用Milli Q超纯水洗涤两边Elispot板子的内部和背面,使用ELISpot读板机检测和分析IFN-γ斑点形成细胞(Spot forming cell,SFC)。
实验结果及结论:游离OVA257-264、GPs和PBS等对照组几乎没有IFN-γ特异性斑点的产生,而GP-OVA257-264免疫则诱导产生了大量的IFN-γ特异性斑点,多达170个/105全脾细胞。证明抗原特异性细胞免疫数量和活性的提升。
实施例9
简称为GP-peptide在体内诱导抗原特异性体液免疫检测(附图9)
将C57BL/6小鼠随机分组,每隔14天在腹股沟用偶联有MHCII限制性模式抗原多肽OVA323-339的GP-OVA323-339疫苗颗粒皮下免疫一次。在第2、3、4次免疫后第7天,用抗凝管从小鼠眼缘静脉采集全血50μL。4℃静置30分钟后,3800rpm离心10分钟,提取上层血清,ELISA检测抗体效价,或置于-80℃冰箱储存。抗体效价的具体检测方法:将10 μg/mL OVA加入96孔ELISA微孔板上,每孔100μL,4℃包被过夜。然后用含0.05% Tween 20的PBS(PBST)缓冲液洗涤4次,并在室温下用5%BSA溶液封闭1小时。用 PBST洗涤4次后,连续梯度稀释血清,并加入到封闭后的孔板中,室温220rpm震荡孵育2 小时。用PBST洗涤4次培养板,向孔板中加入HRP标记的羊抗小鼠IgG、IgG1和IgG2a 抗体,继续在室温下震荡孵育1小时,最后用PBST冲洗5次,加入用TMB单组份显色液显影10-15分钟。待颜色反应足够强,每孔加入100μL的1mol/L的硫酸溶液终止显色反应,并用多功能酶标仪测定各孔在450nm和570nm波长下的吸光度。IgG滴度定义为最大血清稀释度(OD450-OD570)值≥0.5。为了进行统计分析,检测不到信号的IgG、IgG1、 IgG2a滴度统一视作为25。
实验结果及结论:在GP-OVA323-339免疫小鼠中,其IgG,IgG1和IgG2a的滴度都远高于 OVA323-339多肽免疫组的,这些抗体滴度甚至比OVA全蛋白免疫组的还要高,尤其是IgG2a滴度的滴度显著高于OVA全蛋白免疫组的,显示GP偶联多肽的疫苗颗粒免疫会产生明显的Th1偏向性的免疫反应,这使其更有利于抗肿瘤免疫。
实施例10
简称为GP-peptide对小鼠EG7皮下肿瘤模型的抑制效果(附图10)
将C57BL/6小鼠随机分组,每14天在腹股沟皮下免疫三次GP-peptide疫苗颗粒和相应的对照组分子。在第三次免疫接种后7天,将不同数量的肿瘤细胞通过皮下注射接种到小鼠体内在其后背的右侧面(1×106EG7·OVA细胞),然后每2或3天用游标卡尺进行肿瘤最大长度和最大宽度的测量,最后根据“体积=长×宽×宽×0.5”的计算方式计算小鼠肿瘤体积。当肿瘤体积大于1500mm3的小鼠被认为是肿瘤造成的死亡。
实验结果及结论:GP-OVA257-264和PolyI:C、CpG 2395佐剂的联合免疫能够诱导机体产生长期有效的抗肿瘤免疫记忆效应,从而有效抵抗再次的肿瘤入侵。
实施例11
简称为GP-peptide对小鼠B16F10肺转移肿瘤模型的抑制效果(附图11)
将C57BL/6小鼠随机分组,每14天在腹股沟皮下免疫三次连接有M30新抗原多肽的GP-M30疫苗颗粒和免疫佐剂的组合以及相应的对照组疫苗。在第三次免疫接种后7天,对各组小鼠通过静脉注射1×105B16F10黑色素细胞。肿瘤接种后20天后,对小鼠实施安乐死并解剖其完整肺部,对黑色素瘤肺转移灶的数量进行统计。
结果及结论:在PBS对照组中肺肿瘤灶高达57个;而GP-M30联合CpG 2395可显著抑制 B16F10肺转移瘤的形成,只有少于7个肿瘤灶的产生,抑制效率达到了90%;但GP-M30联合PolyI:C的效果却不佳,肿瘤灶也达到了44个。证明GP-M30和CpG 2395佐剂的联合免疫能够诱导机体产生强烈的抗肿瘤转移能力。
实施例12
简称为GP-peptide对小鼠4T1乳腺癌模型的抑制效果(附图12)
将Balb/c小鼠随机分组,每14天在腹股沟皮下免疫三次连接有M25新抗原多肽的GP- M25疫苗颗粒和相应的对照组分子。在第三次免疫接种后7天,将1×1054T1乳腺癌细胞通过皮下注射接种到小鼠体内在其后背的右侧面,然后每2或3天用游标卡尺进行肿瘤最大长度和最大宽度的测量,最后根据“体积=长×宽×宽×0.5”的计算方式计算小鼠肿瘤体积。当肿瘤体积大于1500mm3的小鼠被认为是肿瘤造成的死亡。
实验结果及结论:GP-M25/CpG 2395的组合免疫显示出强大的肿瘤生长抑制效果。在肿瘤接种后19天,M25/CpG 2395或免疫小鼠肿瘤已经大于600mm3;而GP-M25/CpG 2395免疫小鼠的肿瘤体积小于200mm3;不过没有CpG 2395的联合,GP-M25自身的肿瘤免疫抑制效果会降低。
实施例13
简称为GP-peptide对小鼠CT26结肠癌模型的抑制效果(附图13)
取Balb/c小鼠,将5×105CT26结肠癌细胞通过皮下注射接种到小鼠后背的右侧面,然后待肿瘤可见后,给小鼠按照平均肿瘤体积分组(在第五章的肿瘤模型中,为了尝试摸索更好的治疗效果,我们在肿瘤接种后直接对小鼠进行了随机分组,然后在肿瘤接种后第1、 4、8、12天分别对小鼠进行腹股沟皮下免疫四次),然后在肿瘤接种后第5、8、12、17天分别对各组小鼠腹股沟皮下免疫四次连接有ME1和ME4新抗原多肽的GP-ME1-ME4疫苗颗粒和相应的对照组分子。在整个过程中,每2或3天用游标卡尺进行肿瘤最大长度和最大宽度的测量,最后根据“体积=长×宽×宽×0.5”的计算方式计算小鼠肿瘤体积。当肿瘤体积大于 1500mm3的小鼠被认为是肿瘤造成的死亡。
实验结果及结论:在GP-ME1-ME4联合CpG 2395免疫组中,40%的小鼠获得了完全的肿瘤清除,游离ME1、ME4联合CpG 2395免疫,也有20%的小鼠获得了肿瘤的完全清除。ME1和ME4新抗原表位可能具有相对更强的免疫激活能力,依据它构建的治疗性疫苗在肿瘤模型上治疗效果更为明显。与前面的实验结果相一致,三个月后,用5×105CT26细胞对这些肿瘤被清除的小鼠进行再次皮下接种,小鼠100%不会生长肿瘤,
总结:GP-OVA257-264疫苗能够高度靶向抗原递呈细胞、诱导其活化和抗原递呈,并高度激活小鼠抗原特异性细胞免疫,诱导产生了2.8%的抗原特异性CD8 T细胞,并在预防型和治疗性EG7·OVA淋巴瘤模型中均展现了一定的抗肿瘤能力。同时,我们用MHCII限制性的模式新抗原多肽OVA323-339与GPs偶联,得到的GP-OVA323-339颗粒能够诱导强于OVA蛋白的Th1偏向的体液免疫产生。随后,与TLR激动剂PolyI:C和CpG 2395的联合使用大大提高了GP-OVA257-264的抗肿瘤活性,能够在预防性模型中达到33%的完全缓解率。在此基础上,我们在更多的小鼠同系肿瘤模型上进一步验证了GP-peptide的疫苗递送系统的的免疫激活及肿瘤预防和治疗的能力。我们选取了B16F10黑色素瘤、4T1乳腺癌和CT26结肠癌等三种鼠源肿瘤模型,并分别合成他们对应的新抗原多肽,与GPs颗粒偶联,成功构建了针对不同肿瘤的GP-peptide疫苗。我们发现,相比游离的新抗原多肽,几种GP-peptide的接种均能够诱导小鼠产生强烈的新抗原特异性细胞免疫应答,而且GP-peptide与CpG 2395的联合给药在三种新抗原模型中均产生了一定的肿瘤抑制效果。
上述对本发明中涉及的一般性描述和对其具体实施方式的描述不应理解为是对该发明技术方案构成的限制。本领域所属技术人员根据本发明,在不违背所涉及的发明构成要素的前提下,对上述一般性描述或/和具体实施方式(包括实施例)中公开的技术特征进行增加,减少或组合,形成属于所属发明的其它的技术方案,同样在本发明的保护范围。本发明的全部范围由所附权利要求及其任何等同物给出。
Claims (8)
1.一种新型的基于酵母外壳颗粒的新抗原多肽疫苗载体体系(GP-peptide),其设计特征为:将酵母来源的外壳颗粒(GP)通过特定的连接接头与多肽抗原(Peptide)进行偶联。
2.如权利要求1所述的多肽疫苗载体体系,其特征在于,其中所述酵母来源的外壳颗粒含有若干个羟基(OH),并在羟基上连接了含有PEG1的连接接头。
3.如权利要求1、2所述的PEG1中重复单元为-CH2CH2O-;所述重复单元OCH2CH2的个数n=1-10;优选n=3;所述的PEG1其中一端具有结构为与所述酵母来源的外壳颗粒上羟基连接;另一端具有结构为/>与DBCO连接,所述的PEG1由具有如下结构:且由/>反应得到,反应原理如下:
4.如权利要求1所述的多肽疫苗载体体系,其中所述多肽抗原部分独立地选自任意抗原多肽(Peptide),其特征在于,多肽末端含有一个带有巯基(SH)的半胱氨酸(Cys)并在巯基端连接了含有PEG2的连接接头。
5.如权利要求1、4所述的PEG2中重复单元为-CH2CH2O-;所述重复单元OCH2CH2的个数m=1-10;优选m=4;所述的PEG2其中一端具有结构为与所述多肽抗原部分的巯基连接;另一端具有DBCO结构为/>与/>连接,所述的PEG2具有如下结构:
6.制备权利要求1-5中任一项所述的多肽疫苗载体体系的方法(以n=3,m=4为例),其包括:
1)将权利要求1-3中所述PEG1与酵母来源的外壳颗粒(GP)进行连接获得(GP-PEG1);
2)权利要求1、4、5中所述PEG2与多肽抗原(Peptide)进行连接获得(PEG2-Peptide);
3)将获得的GP-PEG1与PEG2-Peptide偶联以获得多肽疫苗载体体系(GP-peptide)。
7.权利要求1-6中任一项所述多肽疫苗载体体系的制备方法,其合成路线为(以n=3,m=4为例):
PEG1的合成
PEG2分子结构
第一步反应
第二步反应
第三步反应
8.权利要求1-6中任一项所述的多肽疫苗载体体系在制备用于预防型疫苗和治疗型疫苗中的用途。
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