CN112245575A - 含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗及其用途 - Google Patents
含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗及其用途 Download PDFInfo
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Abstract
本发明提供了一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，包含至少编码一种肿瘤抗原的mRNA链段和至少一种带正电的支化聚合物大分子，所述的mRNA和支化聚合物大分子复合后被包封在生物相容性脂质双层壳中。本发明以支化聚合物大分子与脂质分子及免疫助剂等多组分构建纳米核壳结构递送针对编码临床肿瘤抗原的mRNA以实现对此类肿瘤的免疫治疗。本发明将可免疫辅助剂与编码mRNA同时整合进入递送疫苗中，并将其有效递送至哺乳动物细胞，以用于治疗或改善哺乳动物中一种或多种癌症的症状。本发明具有制法简便，抗瘤谱广，特异性强，效率高的优点。
Description
技术领域
本发明属于生物医学领域，涉及一种疫苗，具体来说是一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗及其用途。
背景技术
疫苗通常由可以被机体免疫系统识别的一种或者多种抗原及数种佐剂所组成的制剂，通过对人体免疫系统的刺激及激活增强人体对特异性疾病的免疫反应，从而有效抑制该病的发生、发展及复发。疫苗一般分为两类：预防性疫苗和治疗性疫苗。其中，治疗性疫苗在针对包括癌症和急性传染病等威胁生命的多种类型疾病中表现出巨大潜力。
然而，针对癌症的疫苗与针对传染病的产生抗病原体的疫苗完全不同，因为癌症肿瘤内部微环境复杂，效应良好的癌症疫苗必须有效诱导抗肿瘤反应选择的抗原的产生及具有足够的效力以激活肿瘤内部的免疫细胞以杀死肿瘤。目前大多数肿瘤疫苗多使用抗原的多肽片段或表达抗原的DNA质粒作为疫苗的核心成分。蛋白质和肽相对容易制备，并且可以大规模生产，但是，临床中抗原肽的选择主要取决于患者的主要组织相容性复合体(MHC)蛋白的类型，而不同患者的MHC具有极大的差异，且抗原肽往往并非完整的抗原，因而其抗原性及特异性可能与完整抗原有所差异。而DNA疫苗可以快速针对不同表达的抗原构建有效的质粒系统，耗时短并且成本低，然而DNA由于需要进入细胞核以发挥作用，其转录翻译效力低下，并存在不受控制的基因组整合风险。
基于mRNA的癌症疫苗:
近年来，mRNA成为治疗癌症的理想抗原来源疫苗。其保留了DNA疫苗的优点即进行定制以编码多种抗原，且能被表达为完整的抗原，保证了抗原性和特异性，同时可以通过触发细胞Toll样受体(TLR)等充当佐剂抗原呈递细胞中的信号转导。其合成周期也较快，可用于癌症的个体化定制疫苗治疗。此外，mRNA介导的基因转移亦不会诱发宿主细胞基因重组的风险。
但是，由于带负电荷的mRNA分子链段长且分子量较大，难以穿透同样带有负电的抗原呈递细胞。实践中有研究者通过电穿孔的方式将基于患者抗原的mRNA疫苗分子导入患者来源的树突状细胞(DC)，待其表达后将此类培养的DC疫苗重新引入患者体内以获得肿瘤刺激，激活肿瘤的免疫细胞，但是，此类方法需要大量培养自体DC细胞，耗时较长，并不适合大量生产现成的治疗疫苗。
制备mRNA疫苗的另一种方法是包装，将mRNA包封进入纳米粒子中，注入人体后利用人体内抗原呈递细胞对纳米粒子的吞噬作用将抗原表达。这样的例子疫苗是CureVacmRNA疫苗，利用阳离子鱼精蛋白将mRNA通过静电作用压缩至纳米尺度，并将其作为疫苗注射进入患者体内，不仅可以使宿主细胞摄取纳米颗粒，还可以促进宿主细胞中依TLR-7/8信号传导(Scheel等，2005年)。该种疫苗由浓缩在核心中的mRNA分子与鱼精蛋白复合体为主要组成结构(范围从二十纳米至几百纳米)。一旦mRNA疫苗被抗原呈递细胞吸收，mRNA在这些细胞内释放，然后用作产生编码抗原的模板。
现有技术中的缺陷：
此类疫苗的主要问题是：1)mRNA分子部分暴露于体液，因此容易被组织，细胞和/或血浆中所大量存在的核酸酶降解；2)“裸”mRNA分子可以与所有类型的免疫细胞相互作用，可能导致不良的副作用(例如分泌高水平的细胞因子)；3)这种mRNA不能被抗原呈递细胞非常有效地内在化。
由于基于NGS和大数据技术的大规模癌症基因组测序工作的进行和免疫原性肿瘤突变预测技术的进步，开发新的和改良的癌症疫苗的基础已经被有效奠定，然而，肿瘤疫苗的递送技术已经大大落后。
本发明所公开的mRNA纳米递送体系用于肿瘤的免疫治疗代表了新的癌症疫苗的方向。基于本发明公开的mRNA疫苗具有较高的灵活性，可以在同一体系内包含多种新抗原表达的mRNA及免疫佐剂，同时可以基于单个患者的特异性突变谱，构建免疫激活的mRNA片段，使精确治疗或“个性化”疫苗成为可能。
发明内容
本发明的目的在于提供一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗及其用途，所述的这种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗及其用途要解决现有技术中的疫苗抗肿瘤效果不佳的技术问题。
本发明提供了一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，包含至少编码一种肿瘤抗原的mRNA链段和至少一种带正电的支化聚合物，所述的mRNA和支化聚合物大分子复合后被包封在生物相容性脂质双层壳中。
进一步的，带正电聚合物多臂支化聚合物大分子与mRNA所组成的聚电解质为核心，以生物相容性多功能磷脂及胆固醇包裹核心形成脂质双层壳结构。
进一步的，还包括一种或多种癌症治疗剂。
进一步的，还包括一种或多种抗原本体、抗原性多肽或其抗原性肽片段。
进一步的，还包括药学或者生物学上可接受的载体、缓冲液、稀释剂或赋形剂。
本发明还提供了上述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗在制备用于治疗或改善癌症症状的药物中的用途。
进一步的，用于结合mRNA而制备核颗粒的带正电荷的支化聚合物大分子是生物可降解的和/或生物相容的。选自但不限于支化聚胺、支化聚醚、支化聚酯、支化聚脲支化聚砜、支化聚丙烯酸、支化聚丙烯腈、支化聚赖氨酸、支化聚β-氨基酯、支化聚精氨酸、支化聚天冬酰胺支化聚乙烯基亚胺、树枝状PAMAM大分子聚合物及其组合，其支化度包含但不限于三臂、四臂、六臂、八臂、十六臂及其组合，其侧链修饰包含但不限二乙基三胺、三亚乙基四胺、咪唑及其组合。
进一步的，用于制备生物相容性脂质双层壳的磷脂组分包括一种或多种1,2-二油酰基-sn-甘油-3-乙基磷胆碱(EDOPC)、l，2-二油酰基-sn-甘油-3-磷脂酰乙醇胺(DOPE)、l，2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-N-[氨基(聚乙烯乙二醇)-2000](DSPE-PEG)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、O-乙基磷脂酰胆碱(EDPPC)、(2,3-二油酰基-丙基)-三甲胺(DOTAP)的任意组合。
进一步的，脂质组合物(生物相容性磷脂组分)与支化聚合物大分子之间的比例可以是约1∶1、2∶1、3∶1、4∶1、5∶1、6∶1、7∶1、8∶1、9∶1、19∶1、10∶1、11∶1、12∶1、13∶1、14∶1、15∶1(wt/wt)，或这些比例的范围或任一者。
进一步的，所使用的支化聚合物大分子与编码癌症表面抗原的mRNA的氮磷比例可以是10∶1、11∶1、12∶1、13∶1、14∶1、15∶1、16∶1、17∶1、18∶1、19∶1、20∶1、21∶1、22∶1、23∶1、24∶1、25∶1、26∶1、27∶1、28∶1、29∶1、30∶1、31∶1、32∶1、33∶1、34∶1、35∶1、36∶1、37∶1、38∶1、39∶1、40∶1、41∶1、42∶1、43∶1、44∶1、45∶1、46∶1、47∶1、48∶1、49∶1、50∶1、51∶1、52∶1、53∶1、54∶1、55∶1、56∶1、57∶1、58∶1、59∶1、60∶1或这些比例的范围或任一者。
进一步的，编码一种或多种癌症表面抗原的mRNA被配制在纳米核壳结构中，所述纳米粒子具有约10至约100nm，诸如但不限于约10至约20nm、约10至约30nm、约10至约40nm、约10至约50nm、约10至约60nm、约10至约70nm、约10至约80nm、约10至约90nm、约20至约30nm、约20至约40nm、约20至约50nm、约20至约60nm、约20至约70nm、约20至约80nm、约20至约90nm、约20至约100nm、约30至约40nm、约30至约50nm、约30至约60nm、约30至约70nm、约30至约80nm、约30至约90nm、约30至约100nm、约40至约50nm、约40至约60nm、约40至约70nm、约40至约80nm、约40至约90nm、约40至约100nm、约50至约60nm、约50至约70nm约50至约80nm、约50至约90nm、约50至约100nm、约60至约70nm、约60至约80nm、约60至约90nm、约60至约100nm、约70至约80nm、约70至约90nm、约70至约100nm、约80至约90nm、约80至约100nm和/或约90至约100nm的直径。
进一步的，本发明所公开mRNA纳米疫苗组合物具有约10至500nm的直径。在一个实施方案中，所述纳米粒子具有大于100nm、大于150nm、大于200nm、大于250nm、大于300nm、大于350nm、大于400nm、大于450nm、大于500nm、大于550nm、大于600nm、大于650nm、大于700nm、大于750nm、大于800nm、大于850nm、大于900nm、大于950nm或大于1000nm的直径。
进一步的，本发明所公开mRNA纳米疫苗组合物可以是相对均匀的。其多分散性指数有约0至约0.25，如0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24或0.25的多分散性指数。在一些实施方案中，本文公开的纳米粒子组合物的多分散性指数可以是约0.10至约0.50。
进一步的，所述的疫苗还包括一种或多种佐剂，其包括但不限于CpG寡核酸、poly(I/C)、环状GMP-AMP(cGAMP)、脂多糖(LPS)、单磷酰脂质A(MPLA)、明矾或其任何组合。各种佐剂可有效包裹在mRNA核心内或核心颗粒和包封的亲水性磷脂双层之间的空间之中。
进一步的，本发明所公开疫苗组成还可进一步结合一种或多种治疗剂，其可包括(但不限于)一种或多种化合物，例如顺铂、奥沙利铂、5-氟尿嘧啶、环磷酰胺、阿霉素、吉西他滨、多西他赛、紫杉醇、曲妥珠单抗、甲氨蝶呤、卡铂、长春瑞滨、卡培他滨、伊利替康、阿替利珠、德瓦鲁、米托蒽醌、伊沙贝比隆、阿利库、艾瑞布林、拉帕替尼、卡莫斯汀、氮芥末、硫芥末、长春新碱，喜树碱或其任何组合。
进一步的，所制备疫苗可以进一步包括一种或多种抗原本体，抗原性多肽或其抗原性肽片段，包括由细胞分别产生的多种抗原肽，可根据抗原肽的水溶性不同将不同抗原肽有效包裹在mRNA核心内或核心颗粒和包封的亲水性磷脂双层之间的空间之中。
进一步的，本发明所公开的癌症疫苗递送组合物可将其与一种或多种药学或者生物学上可接受的载体，缓冲液，稀释剂或赋形剂，如脂质体的混合物、脂质体、磷脂、鞘脂或其他细胞自身产生的囊泡类型所结合提高生物相容性与递送效率。
进一步的，本发明所公开的癌症疫苗递送组合物将用于哺乳动物全身性给药，如皮下或静脉内给药，肌肉给药，腹膜给药，淋巴结给药或眼内给药，口服或以其他形式的皮肤贴剂。注射后疫苗通过接触和吞噬作用于一种或多种抗原呈递细胞，包括但不限于人树突状细胞细胞，巨噬细胞和B细胞，以激活抗肿瘤免疫原性。
进一步的，本发明所公开的癌症疫苗递送组合物将用目标优选于在特定的细胞类型中表达，如APC、DC或/和T细胞。所激活的APC、DC或/和T细胞的目标表达相对于对照群体，即包含非激活的APC、DC或/和T细胞来说至少为2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、20倍、50倍、100倍、500倍。
进一步的，本发明所公开的癌症疫苗递送组合物可向B细胞有效呈递抗原，目标为靶向B细胞标记物的mRNA序列，包括在B细胞中及B细胞表面表达的抗原序列，通过在纳米载体表面的结构修饰及支化聚合物大分子的结构调节优化抗原呈递效率，结构上完整的免疫调节剂以高拷贝数目及密度通过癌症疫苗递送组合物向B细胞呈递。
进一步的，B细胞靶向可以是伴随的由结合补体受体CR1的部分形成。在某些实施例中，B细胞靶向可通过B细胞表面标记作为CD19，CD20和/或CD22。在一些实施方案中，B细胞可以通过B细胞标记(例如CD40，CD52，CD80，CXCR5，VLA-4，II类MHC，表面IgM或IgD，APRL和/或BAFF-R。本发明涵盖了同时定位B的认识补体受体或其他特异部分的细胞APC相关分子可增强体液反应。
进一步的，B细胞靶向可以是伴随的由与任何实体(例如蛋白质，脂质，碳水化合物，小分子等)特异性结合的任何靶向部分形成在B细胞(即B细胞标记)。示例性的B细胞标记包括但不仅限于(排序不分大小),CD1c(M241,R7)；CD1d(R3)；CD2(E-rosette R,T11,LFA-2)；CD5(T1,Tp67,Leu-1,Ly-1)；CD6(T12)；CD9(p24,DRAP-1,MRP-1)；CD11a(LFA-1C,CLIntegrinchain)；CD11b(CM Integrin chain,CR3,Mo1,C3niR,Mac1)；CD11c(OX Integrin,P150,95,AXb2)；CDv17(Lactosylceramide,Lac-Cer)；CD18(Integrin B2,CD11a,b,c C-subunit)；CD19(B4)；CD20(B1,Bp35)；CD21(CR2,EBV-R,C3dR):CD22(BL-CAM,Lyb8,Siglec-2)；CD23(Fell RII,B6,BLAST-2,Leu-20)；CD24(BBA-1,HSA)；CD25(Tac antigen,IL-2RC,p55)；CD26(DPP IV Dectoeneyme,ADAbinding protein)；CD27(T14,S 152)；CD29(Platelet GPIIa,B-1integrin,GP)；CD31(PECAM-1,Endo-Cam)；CD32(FCYRII)；CD35(CR1,C3b/C4b receptor)；CD37(gp52-40)；CD38(ADP-ribose cyclase,T10)；CD39(ATPdehydrogenase,NTPdehydrogenase-1)；CD40(Bp50):CD44(ECMRII,H-CAM,Pgp-1)；CD45(LCA,T200,B220,Ly5)；CD45RA:CD45RB:CD45RC:CD45RO(UCHL-1)；CD46(MCP):CD47(gp42,IAP,OA3,Neutrophilic):CD47R(MEM-133)；CD48(Blast-1,Hulym3,BCM-1,OX-45)；CD49b(VLA-2C.,gpla,C2 Integrin)；CD49c(VLA-3C,O.3Integrin):CD49d(VLA-4C,C4Integrin)；CD50(ICAM-3)；CD52(CAMPATH-1,HES)；CD53(OX-44)；CD54(ICAM1)；CD55(DAF)；CD58(LFA-3)；CD60a(GD3)；CD62L(L-selectin,LAM-1,LECAM-1,MEL-14,Leu8,TO1):CD72(Ly-19.2,Ly-32.2,Lyb-2)；CD73(Ecto-5'-nuciotidase)；CD74(Ii,invariant chain)；CD75(sialo-masked Lacto amine)；CD75S(C2,6salivatedLacto amine)；CD77(PKantigen,BLA,CTH/Gb3)；CD79a(IgC,MB1)；CD79b(Igf3,B29)；CD80；CD81(TAPA-1)；CD82(4F9,C33,IA4,KAI1,R2)；CD83(HB15)；CD84(P75,GR6)；CD85(ILT2,LIR1,MIR7)；CDw92(p70)；CD95(APO-1,FAS,TNFRSF6)；CD98(4F2,FRP-1,RL-388)；CD99(MIC2,E2)；CD100(SEMA4D)；CD102(ICAM-2)；CD108(SEMA7A,JMHblood group antigen)；CDw119(IFNYR,IFNYRa)；CD120a(TNFRI,p55)；CD120b(TNFRII,p75,TNFR p80)；CD121b(Type 2IL-1R):CD122(IL2RB)；CD124(IL-4RC)；CD130(gp130)；CD132(Common Y chain,IL-2Ry)；CDw137(4-1BB,ILA)；CD139；CD147(Basigin,EMMPRIN,M6,OX47)；CD150(SLAM,IPO-3)；CD162(PSGL-1)；CD164(MGC-24,MUC-24)；CD166(ALCAM,KG-CAM,SC-1,BEN,DM-GRASP):CD167a(DDR1,trkE,Cak)；CD171(L1CMA,NILE)；CD175s(Sallyl-Tn(S-Tn))；CD180(RP105,Bgp95,Ly64)；CD184(CXCR4,NPY3R):CD185(CXCR5)；CD192(CCR2)；CD196(CCR6)；CD197(CCR7(wasCDw197))；CDw197(CCR7,EBI1,BLR2)；CD200(OX2)；CD205(DEC-205)；CDw-10(CK)；CD213a(CK)；CDw-17(CK)；CDw-18a(IL18RO)；CDw-18b(IL18Rf8)；CD220(Insulin R):CD221(IGF1R):CD222(M6P-R,IGFII-R):CD224(GGT):CD225(Leu13)；CD226(DNAM1,PTA1)；CD227(MUC1,PUM,PEM,EMA)；CD229(Ly9)；CD230(Prion Protein(Prp))；CD232(VESP-R):CD245(p220/240)；CD24？(CD3 Zeta Chain)；CD261(TRAIL-R1,TNF-R superfamily,member 10a).CD262(TRAIL-R2,TNF-R Superfamily,member 10b)；CD263(TRAIL-R3,TNF-R superfamily,member 10c).CD264(TRAIL-R4,TNF-R Superfamily,member 10d)；CD265(TRANCE-R,TNF-Rsuperfamily,member 11a)；CD267(TACI,TNF-R superfamily,member 13B)；CD268(BAFFR,TNF-R superfamily,member 13C)；CD269(BCMA,TNF-Rsuperfamily,member 16)；CD275(B7H2,ICOSL):CD277(BT3.1.B7 family:Butyrophilin3)；CD295(LEPR):CD298(ATP1B3Na-KATPase B3 subunit)；CD300a(CMRF-35H):CD300c(CMRF-35A)；CD305(LAIR1)；CD307(IRTA2)；CD315(CD9P1)；CD316(EW12)；CD317(BST2)；CD319(CRACC,SLAMF7)；CD321(JAM1)；CD322(JAM2)；CDw327(Siglecó,CD33L):CD68(gp100,Macrosialin):CXCR5；VLA-4；classII MHC；其中，在括号中列出的名称代表替代名称。
进一步的，B细胞靶向可以是伴随的由与任何靶标特异性结合的任何靶向部分形成实体(例如蛋白质，脂质，碳水化合物，小分子等)在B细胞上突出表达和/或存在的多重细胞激活因子(即激活的B细胞标记)。示例性激活B细胞标记包括但不限于CD1a(R4，T6，HTA-1)；CD1b(R1)；CD15(Sally Lewis X)；CD15u(3'硫磺路易斯酸)；CD15su(6-Sulpho-SialylLewis X)；CD30Ber-H2，Ki-1)；CD69(AIM，EA1，MLR3，gp34/28，VEA)：CD70(Ki-24，CD27配体)；CD80(B7，B7-1，BB1)；CD86(B7-21B70)；CD97(BL-KDD/F12)；CD125(IL-5Ro)；CD126(IL-6R.C。)；CD138(Syndecan-1，硫酸乙酰肝素，糖聚糖)；CD152(CTLA-4)；CD252(OX400L，TNFsuperfamily,member4)；CD253(TRAIL，TNFsuperfamily,member10)；CD279(PD1)；CD289(TLR9，Toll样受体9)；和CD312(EMR2)；其中括号中列出的名称代表备用名称。
进一步的，本发明所公开的mRNA的疫苗组合物是用于治疗或改善动物的一种或多种癌症症状。在一些实施方案中，癌症可以是诊断为或鉴定为难治性，转移性，复发性或复发性耐药治疗的癌症。此类癌症的例子包括但不限于乳腺癌，肺癌癌症，大肠癌，胃癌，胰腺癌，胶质母细胞瘤，头颈癌症，白血病，淋巴瘤，多发性骨髓瘤，肝癌，肾癌，膀胱癌，黑色素瘤和相关疾病。在某些实施方案中，癌症可以是转移性癌症，例如转移性乳腺癌，转移性肺癌，转移性黑色素瘤或转移性结直肠癌癌症，胃癌，胰腺癌，头颈癌，肝脏癌症，肾癌，膀胱癌或受影响的一种或多种相关疾病。
本发明所公开的mRNA的疫苗组合物可以进一步调整施用药物的步骤、联合有效量的放射线或其他化学疗法共同治疗，可以一次给药，也可以连续多次给药一两天以上，一周或几周的时间或一个或多个月或更长时间。
本发明所公开的mRNA的疫苗组合物可应用于各种体外，离体，和体内治疗方案，它们可以单独配制，或者与一种或多种其他试剂的组合，包括但不限于一种或多种更多抗癌抗原，一种或多种抗原肽，一种或多种诊断剂试剂，一种或多种治疗剂，一种或多种细胞毒性试剂，一种或多种化学治疗剂，一种或多种佐剂，一种或多种免疫刺激剂，一种或多种免疫调节剂，或其任何组合，用于多种治疗适应症，包括但不限于治疗或改善一种或多种人类癌症，过度增殖性疾病，传染性疾病的症状疾病及心脏病等。
本发明所公开的mRNA的疫苗组合物可以进一步联合包括一种或多种活性剂，例如一种或多种预防剂，一种或多种更多治疗剂，一种或多种诊断剂，一种或多种疫苗，一种或多种成像剂，一种或多种放射性标记，一种或多种佐剂，一种或更多化学治疗剂，一种或多种细胞毒性剂，一种或多种免疫检查点抑制剂药物，例如抗PD-1抗体、抗PD-L1抗体、抗CTLA-4抗体等，或其任何组合。
本发明所公开的mRNA的组合物在某些应用中可用于将受试患者的免疫原性细胞体外刺激培养，然后随后将所得的免疫激活细胞重新引入主体。特别考虑到这种离体疗法可用于将有效的mRNA抗原疫苗引入人类树突状细胞，使活性成分与细胞接触，然后将转化细胞扩增并重新输回动物体内以实现免疫治疗。
在特定的实施方案中，本发明所公开的mRNA疫苗的组合物可以于体外扩增群体肿瘤抗原特异性T细胞。如共培养源自人类患者的T细胞，以扩展肿瘤抗原特异性的T细胞并输回患者体内杀灭抗原表达细胞。
另外，本发明所公开的mRNA疫苗的组合物也可以用于分离肿瘤用于T细胞工程的抗原特异性T细胞受体。一个例子是mRNA疫苗处理后的树突状细胞或B细胞与人T细胞共培养，从而识别并分离出T细胞由此具有高的结合能力。一旦分离出T细胞，它们的T细胞受体可以通过抗原预测技术测序确定，并用于产生TCR-T细胞(另一个癌症免疫疗法的分支)。
本发明的疫苗可向一种或多种哺乳动物细胞，如人树突状细胞，人巨噬细胞和人B细胞等呈递抗肿瘤抗原。
本发明所述即使用支化聚合物大分子复合mRNA并使用脂质体将复合物包裹的方法构建稳定的mRNA递送体系，通过对抗原提呈细胞(例如树突状细胞)中的进行基因递送的方法使其表面表达相应抗原及免疫应激信号分子，并有效激活抗癌免疫细胞进而治疗癌症。
本发明所公开的支化聚合物大分子/脂质分子所构成的多层纳米组合结构可以有效地递送多种核酸分子(包括，例如，编码一个或更多的癌症或肿瘤特异性抗原的mRNA、DNA、siRNA等)。通过阳离子支化聚合物大分子的静电复合作用将大分子核酸包装在“核心”结构中，随后使用两亲性的脂质分子将该“核心”结构包裹于脂双层的“壳”中。该多层纳米组合结构可将抗原表达基因传递到一个或多个选定的哺乳动物细胞，例如但不限于一种或多种抗原呈递细胞，如树突状细胞、淋巴细胞、巨噬细胞和B细胞等。支化聚合物大分子与脂双层结构的存在可有效保护抗原呈递核酸分子被体液中的核酸酶降解。此外，亲脂性壳层结构还提供了通过抗原呈递细胞的有效通道，并可通过调节其组成成分以更加有效的激活抗原呈递细胞，可表现出更强的抗肿瘤免疫效果。
进一步的，本发明公开提供了核-壳结构比传统的mRNA纳米疫苗表现出更有效的免疫响应因子INF-γ和TNF-α的刺激作用。本发明基于多组分mRNA的肿瘤抗原编码治疗疫苗可用于治疗原发哺乳动物的一种或多种疾病，特别是用于治疗和改善一种或多种的哺乳动物癌症的症状。
本发明公开了以支化聚合物大分子与脂质分子及免疫助剂等多组分构建纳米核壳结构递送针对编码临床肿瘤抗原的mRNA以实现对此类肿瘤的免疫治疗。本发明所公开的结构配方中将可免疫辅助剂与编码mRNA同时整合进入递送疫苗中，并将其有效递送至哺乳动物细胞，以用于治疗或改善哺乳动物中一种或多种癌症的症状。本发明具有制法简便，抗瘤谱广，特异性强，效率高的优点。
附图说明
为了促进对本发明的原理的了解，现在将参考实施例，或者图示和特定语言中的示例用于描述本发明。参照附图可以更好地理解本发明。结合附图进行的以下描述，其中相同的参考数字表示相同的元素，其中：
图1显示了本发明所公开的mRNA疫苗组合物的基本机构与制备过程。图1A是基于支化聚合物大分子-mRNA所组成核心的示意图，通过带正电的支化聚合物大分子与带负电荷的mRNA分子复合，将得到支化聚合物大分子-mRNA封装核心。图1B显示了利用NanoassembelerTM将支化聚合物大分子-mRNA核心用脂质分子包覆，构成本发明所展示的mRNA疫苗组合物核壳结构。
图2显示了对本发明所公开的mRNA疫苗组合物的纳米结构的基本表征，图2A利用凝胶阻滞的方法显示了示例性支化聚合物大分子与mRNA的在不同重量比条件下的结合能力。图2B，2C显示了支化聚合物大分子/mRNA核心在不同重量比条件下的核心的粒径及表面电荷的变化。随着不同比例脂质分子的加入，mRNA疫苗组合物的粒径与表面电荷也出现了一定的变化(图2D)；并对抗原呈递树状细胞DC2.4表现出了较高的转染效率(图2E)。
图3显示了示例性的mRNA疫苗组合物对mRNA的复合效率与生物毒性。如图3A所示，纳米核壳结构对mRNA的包封率达到了95％以上，同时也没有表现出明显的细胞毒性(图3B)。
图4显示了mRNA疫苗组合物的抗肿瘤活性与活体毒性。C57小鼠在第0，3，8天分别注射整合编码OVA蛋白的mRNA疫苗组合物，两周后将表达OVA的E.G7小鼠淋巴瘤細胞系与皮下注入(图4A)，并检测肿瘤生长与小鼠体重变化，可以看出mRNA疫苗组合物有效抑制了肿瘤的生长并没有表现出明显的体重下降(图4B、4C)。
如图5显示了与未接种疫苗的小鼠相比，mRNA疫苗组合物治疗组中的小鼠脾、淋巴结和外周血中的CD8+T细胞显着增加(图5A)；同时如图5B所示，mRNA疫苗组合物治疗组中的表明免疫反应发生的关键指标即来自CD4+T细胞和CD8+T细胞的细胞因子(包括IFN-γ和TNF-α)亦有明显升高。
具体实施方式：
本发明公开了一种以生物相容性支化聚合物大分子、脂质分子及免疫助剂等多组分所构建的纳米核壳结构递送针对临床肿瘤抗原编码的mRNA用于治疗或改善哺乳动物中一种或多种癌症的症状。
下面通过具体实施例对本发明进行详细和具体的介绍，以使更好的理解本发明，但是下实施例并不限制本发明范围。
实验材料及实验动物：
PAMAMG0、二甲基甲酰胺、二乙烯三胺、3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐购自Sigma-Aldrich(美国威斯康星州密尔沃基市)；天冬氨酸苄酯环状酸酐购自Nanosoft Polymer(美国北卡罗莱纳州温斯顿市)；脂质分子(2,3-二油酰基-丙基)-三甲胺、二棕榈酰磷脂酰胆碱及胆固醇购自AvantiPolarLipids Co.(美国阿拉巴马州斯特尔市)；表达虫荧光素酶、绿色荧光蛋白(GFP)及卵清蛋白的mRNA购自
Biotechnologies(美国加利福尼亚圣地亚哥市)。
实施例中所使用的E.G7-OVA和DC2.4细胞(购自美国弗吉尼亚州马纳萨斯市的美国典型培养物保藏中心)，细胞在含有10％(v/v)胎牛血清(FBS，上海ExCell Bio，Inc.，中国上海)的Dulbecco改良的Eagle培养基(DMEM，Life Technologies，Eggenstein，德国)中培养，在37℃恒温箱和5％(体积百分比)CO2水平下，每三个月检查一次细胞是否存在支原体污染。
实施例1：支化聚合物大分子的构建：
将0.2g多臂引发剂(如PAMAMG0)溶解于干燥的100ml二甲基甲酰胺中，并加入10g天冬氨酸苄酯环状酸酐与室温搅拌48小时后使用乙醚将产物沉淀。为获得侧链带有阳离子的支化聚合物大分子，可将所获得的支化聚氨基酸脱保护，具体为将1g支化聚合物大分子溶解于10ml的二甲基亚砜中，并向其中滴加50倍比侧基的二乙烯三胺，室温反应1小时并将使用盐酸将溶液的pH值调节为2.0，随后用蒸馏水透析并冻干以获得阳离子支化聚合物大分子(具体反应过程及结构如下所示)。
实施例2：支化聚合物大分子与mRNA的复合：
琼脂糖凝胶电泳：将所合成的支化聚合物大分子与编码卵清蛋白(OVA)的mRNA链段溶于蒸馏水并依照不同的氮磷比进行混合并在室温下稳定15分钟(图1A)，随后将复合物加入质量百分比浓度为2％的琼脂糖凝胶中80V电压运行30分钟，随后将凝胶使用溴化乙锭染色并使用ChemDocTM MP凝胶成像系统(Bio-Rad)对之进行成像。结果如图2A所示，在氮磷比大于1时mRNA被支化聚合物大分子完全包裹。
粒度电位：将所合成的支化聚合物大分子与mRNA溶于蒸馏水并依照不同的氮磷比进行混合并在室温下稳定15分钟，随后使用MalvernNanosizer ZS测定不同粒子的粒径大小与表面电位。所得结果如图2B，2C所示，支化聚合物大分子与mRNA在不同氮磷比条件下所形成的纳米粒子的粒径由40nm到100nm不等，电位由－40mV至20mV。
实施例2：核壳结构mRNA纳米疫苗的构建：
如图1B所示，将依照不同氮磷比制备的支化聚合物大分子与mRNA复合后的纳米粒子分散在无RNA酶的水中，制为溶液A，定容至0.1ml至10ml，mRNA浓度1μg/ml至100μg/ml；将脂质分子如(2,3-二油酰基-丙基)-三甲胺、二棕榈酰磷脂酰胆碱及胆固醇的乙醇溶液按不同比例混合，制为溶液B，最终定容至0.1ml到10ml。使用NanoassemblrTM将溶液A与溶液B混合，混合流速由0.1ml/min至10ml/min。制备后使用超滤离心除去体系残存乙醇，即得到核壳结构mRNA纳米疫苗。
用上述方法所获得的核壳结构mRNA纳米疫苗的表面电位与粒度同样使用MalvernNanosizer ZS测定，结果如图2D所示，当支化聚合物大分子与mRNA的氮磷比由5至30时，不同比例(2,3-二油酰基-丙基)-三甲胺、二棕榈酰磷脂酰胆碱及胆固醇与之混合所得到的核壳结构mRNA纳米疫苗的粒度可由40nm至80nm不等，电位在20mV至40mV之间。
所制备的核壳结构mRNA纳米疫苗的细胞转染效率使用小鼠骨髓树突状细胞DC2.4细胞测定，具体方法如下：以每孔10000个细胞的密度在96孔板中种入DC2.4细胞，并在37℃，5％(体积百分比)二氧化碳条件下培养24小时。将编码萤火虫荧光素酶的mRNA照前述方法复合为不同氮磷比及不同脂质分子比的核壳结构mRNA纳米制剂(mRNA浓度为0.1μg/ml)，并将制剂加入孔板与DC2.4细胞共培养48小时，随后将细胞裂解并使用Promega的荧光素酶测定试剂盒对细胞中所表达的荧光素酶进行定量分析。所得结果如图2E所示，在(2,3-二油酰基-丙基)-三甲胺、二棕榈酰磷脂酰胆碱及胆固醇的比例为40：20：40的情况下，所制备的核壳结构mRNA纳米制剂对免疫细胞的转染效率都在80％以上。
实施例3：核壳结构对mRNA包裹效率及体外毒性评估：
将依照不同氮磷比制备的支化聚合物大分子与已知浓度的mRNA复合的纳米粒子分散在无RNA酶的水中，制为溶液A，定容至0.1ml至10ml，mRNA浓度1μg/ml至100μg/ml；将脂质分子如(2,3-二油酰基-丙基)-三甲胺、二棕榈酰磷脂酰胆碱及胆固醇的乙醇溶液上述最优比例40:20:40(wt:wt)混合，制为溶液B，最终定容至0.1ml到10ml。使用NanoassemblrTM将溶液A与溶液B混合，混合流速由0.1ml/min至10ml/min。制备后使用超滤离心收集滤液，滤液中的残余mRNA使用Ribo Green试剂盒进行测定，mRNA的包裹效率计算公式如下：
所得结果如图3A所示，通过不同氮磷比与脂质分子混合所制备的核壳结构纳米粒子对外源性mRNA的包裹效率皆在95％以上。
核壳结构mRNA纳米疫苗对细胞的毒性使用MTT的方法测定，其具体实施方法如下：
1、将DC2.4种植于96孔板中，密度为8000细胞/孔，37℃孵育24小时。
2、将制备的核壳结构mRNA纳米疫苗以不同mRNA浓度加入到96孔板中，与细胞共培养72h。
3、在孔板中加入10μl的MTT溶液(5mg/ml)，37℃与细胞共孵育4小时。
4、将培养基倾倒，在96孔板中加入150μl的二甲基亚砜，室温震荡5分钟。
5、使用Bio-Rad酶标仪测定孔板在570nm处的吸收值。
核壳结构mRNA纳米疫苗对细胞的毒性的评估公式如下：
所得结果如图3B所示，通过不同氮磷比与脂质分子混合所制备的核壳结构mRNA纳米粒子在正常的转染浓度内(≤0.5μg/ml)对小鼠骨髓树突状细胞DC2.4并未表现出明显毒性。同时，纳米粒子的细胞毒性呈现出一定的氮磷比依赖性，高的氮磷比表现出了较高的毒性，考虑到在低氮磷比条件下所制备的核壳结构mRNA纳米粒子与高氮磷比条件下所制备的核壳结构mRNA纳米粒子表现出相似的转染效率与包裹效率(图2E、图3A)，在产品的活体使用中，可以使用低氮磷比的mRNA纳米疫苗以在保证免疫细胞激活的同时降低非特异性毒性。
实施例4：核壳结构mRNA纳米疫苗的肿瘤抑制效应评估：
通过最优化比例(2,3-二油酰基-丙基)-三甲胺、二棕榈酰磷脂酰胆碱及胆固醇的比例为40：20：40(wt:wt)；支化聚合物大分子与mRNA核心氮磷比为10，制备包裹OVAmRNA的核壳结构mRNA纳米疫苗，并对C57小鼠进行尾静脉注射，注射剂量为40μgmOVA每公斤。具体操作方案如图4A所示，在第0、3和8天分别给药三次，同时使用接受PBS溶液和无mRNA的核壳结构纳米粒子注射的小鼠设为对照。在接受最后一次免疫注射后的第五天，将3.0×105个E.G7-OVA肿瘤细胞注入小鼠侧面皮下，并每三天通过用卡尺测量垂直直径来监测肿瘤的生长。使用以下公式计算估计的肿瘤体积：长×宽2/2，同时记录小鼠的体重。
本发明所公开的核壳结构mRNA纳米疫苗的肿瘤抑制活性通过对上述荷E.G7OVA淋巴肿瘤的C57小鼠模型表征。结果如图4B所示，注射PBS及空白核壳纳米粒子的小鼠长出了快速生长的肿瘤，而与之相反，注射含OVA mRNA的核壳结构mRNA纳米疫苗的肿瘤生长被有效抑制，相较于PBS及空白纳米粒子组，该类肿瘤的体积仅相当于前者的45.7％，显示出了良好的抗肿瘤免疫活性。与此同时，空白的核壳结构纳米粒子与mRNA纳米疫苗均未显示出明显的动物毒性，即所注射的老鼠的体重没有出现显著的下降(图4C)
实施例5：核壳结构mRNA纳米疫苗的体内免疫效应评估：
在上述实验中，当动物表现出健康受损的迹象或肿瘤的长度超过15毫米时，对动物实施二氧化碳安乐死，并收集其脾脏，腹股沟淋巴结和血液，进行消化粉碎处理并用荧光标记的抗小鼠CD8(BioLegend，Inc.，美国圣地亚哥)和OVA MHC I类四聚体进行染色，然后通过BD FACSVerseTM流式细胞仪进行分析。
为了检测组织内分泌的细胞免疫因子(如IFN-γ和TNF-α)，使用含布雷菲德菌素A的细胞活化鸡尾酒试剂盒(BioLegend，Inc.，圣地亚哥，美国)对所提取组织内淋巴细胞进行刺激，并用T细胞特异性抗体染色。然后对组织与细胞进行固定，使用Triton X-100对其进行通透后，用抗细胞因子抗体对细胞进行染色。表达的细胞免疫因子使用BD FACSVerseTM流式细胞仪(BD Biosciences，贝德福德，美国)获取流式细胞仪数据，并使用FlowJo V10软件进行分析。
所得结果如图5所示，与未接种疫苗的小鼠相比，接种有OVA mRNA的核壳结构mRNA纳米疫苗的小鼠的脾，淋巴结和外周血中CD8+Tetramer+T细胞显着增加(分别为4.3％对6.35％，3.18％对7.82％和0.88％对2.54％)(图5A)。同时，经OVA mRNA的核壳结构mRNA纳米疫苗处理的小鼠CD4+T细胞和CD8+T细胞分泌的细胞因子(包括IFN-γ和TNF-α)相较PBS组与空白纳米粒子组有了显著的增加(图5B)，说明了OVA mRNA的核壳结构mRNA纳米疫苗对小鼠免疫系统的有效激活与增强小鼠的免疫记忆功能，上述结果证明了OVA mRNA的核壳结构mRNA纳米疫苗对免疫抗肿瘤的有效性。
实施例总结：
本发明通过构建含脂质分子、支化聚合物大分子与针对肿瘤表面抗原的mRNA的核壳结构抗肿瘤纳米疫苗，用于癌症免疫基因治疗。实施例结果表明，此类核壳结构所构建的纳米疫苗可以高效包裹mRNA，有效的转染免疫组织中的DC细胞而不引发细胞毒性。
动物实施例表明，本发明所公开的核壳结构抗肿瘤纳米疫苗在侵袭性E.G7-OVA淋巴瘤模型中通过有效激活机体免疫活性从而显示出较强的肿瘤预防作用。因此，本发明所公开的核壳结构抗肿瘤纳米疫苗是mRNA疫苗的合适平台，并具有广泛的临床转化前景。
Claims (10)
1.一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：包含至少编码一种肿瘤抗原的mRNA链段和至少一种带正电的支化聚合物，所述的mRNA和支化聚合物大分子复合后被包封在生物相容性脂质双层壳中。
2.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：带正电的聚合物多臂支化聚合物大分子与mRNA所组成的聚电解质为核心，以生物相容性多功能磷脂及胆固醇包裹核心形成脂质双层壳结构。
3.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：还包括佐剂，所述的佐剂为CpG寡核酸、poly、环状GMP-AMP、脂多糖、单磷酰脂质A或者明矾中的任意一种或者两种以上的组合。
4.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：所述的支化聚合物为支化聚胺、支化聚醚、支化聚酯、支化聚脲、支化聚砜、支化聚丙烯酸、支化聚丙烯腈、支化聚赖氨酸、支化聚β-氨基酯、支化聚精氨酸、支化聚天冬酰胺支化聚乙烯基亚胺或者树枝状PAMAM大分子聚合物中的任意一种或者两种以上的组合。
5.根据权利要求4所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：所述的支化聚合物大分子的支化度为三臂、四臂、六臂、八臂或者十六臂，所述的支化聚合物大分子的侧链修饰为二乙基三胺、三亚乙基四胺或者咪唑。
6.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：所述的生物相容性脂质双层壳的磷脂组分为1,2-二油酰基-sn-甘油-3-乙基磷胆碱、l，2-二油酰基-sn-甘油-3-磷脂酰乙醇胺、l，2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-N-[氨基（聚乙烯乙二醇）-2000]、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺、O-乙基磷脂酰胆碱、(2,3-二油酰基-丙基)-三甲胺中的任意一种或者两种以上的组合。
7.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：还包括一种或多种癌症治疗剂。
8.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：还包括一种或多种抗原本体、抗原性多肽或其抗原性肽片段。
9.根据权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗，其特征在于：还包括药学或者生物学上可接受的载体、缓冲液、稀释剂或赋形剂。
10.权利要求1所述的一种含支化聚合物和mRNA的核壳结构的抗肿瘤疫苗在制备用于治疗或改善癌症症状的药物中的用途。
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