CN104066443A - 用于肿瘤免疫疗法的疫苗 - Google Patents
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Abstract
本发明涉及用于肿瘤免疫疗法的包含树突细胞和细菌菌影的疫苗。
Description
本发明涉及用于肿瘤免疫疗法的包含树突细胞和细菌菌影(bacterial ghosts)的疫苗。
基于树突细胞的疗法使用肿瘤细胞来刺激抗肿瘤免疫力。因此,用肿瘤细胞,特别地用自体肿瘤细胞(即患者自己的肿瘤细胞)温育树突细胞来刺激抗肿瘤免疫力。与肿瘤细胞温育的树突细胞直接从所述肿瘤细胞提取抗原。随后将具有肿瘤相关抗原的树突细胞用作疫苗来刺激免疫***抗肿瘤。虽然树突细胞是活化抗癌反应所必需的,但在无先前活化的情况下它们通常是无效的,因为它们不能将正在生长的癌症识别为危险。通过使用外部刺激活化树突细胞,可产生呈递相关的肿瘤相关抗原、从而诱导有效抗肿瘤反应的树突细胞。
这样的方法描述于例如U.S.2008/0031900中。在其中,在一种或多种癌细胞存在的情况下用GM-CSF和干扰素α活化抗原呈递细胞例如树突细胞。
虽然对于这样的组合物已取得相当大的进展,但仍然需要进一步的改善。
因此,本发明提供了组合物,其包含:
(i)抗原呈递细胞(APC),
(ii)肿瘤相关抗原(TAA),和
(iii)细菌菌影(BG)。
根据本发明,已发现抗原呈递细胞,特别地树突细胞作为肿瘤疫苗的功效可通过提供额外地包含细菌菌影的组合物来改善。
细菌菌影(BG)是细菌(特别地革兰氏阴性细菌)的空细菌细胞包膜。优选的细菌是大肠杆菌(E.coli)或弗氏志贺菌2a(Shigella flexneri2a)或溶血曼海姆菌(Mannheimia haemolytica),特别地大肠杆菌Nissle1917。
BG可通过引起细菌膜完整性破坏、从而导致细菌裂解的异源基因的受控表达来产生。裂解基因的一个实例是噬菌体PhiX174基因E,其编码触发细菌细胞的内膜与外膜融合并形成横跨整个细胞包膜的跨膜通道结构的多肽,通过所述通道结构,整个细胞质内容物因细胞内部与培养基之间的渗透压的变化而被排出,同时内膜和外膜结构得到保留并且保持完整(参照U.S.7,968,323B2)。跨膜通道结构的大小取决于裂解条件,并且内膜直径在20-400nm的范围内。BG的空体不存在核酸、核糖体和其它组分,而基本的内膜和外膜结构(包括抗原性分子,例如外膜蛋白、粘附蛋白(adhesins)、脂多糖(LPS)和肽聚糖)是非变性的并且保持完整。绝对不存在在受控裂解过程的诱导后回复为病原性形式的风险。
细菌菌影可通过包括下列步骤的方法来制备:
(a)提供包含编码能够在细菌细胞包膜中形成通道结构的裂解蛋白的基因的革兰氏阴性细菌细胞;
(b)任选地在其中裂解基因不表达的条件下培养所述细菌细胞;
(c)将细菌细胞经历其中所述裂解基因发生表达并且释放细菌细胞的细胞质组分的条件;和
(d)获得所得的细菌菌影。
编码裂解蛋白的基因的一个优选实例是噬菌体phiX174基因E。
特别优选的,用于上述细菌菌影制备的方法中的细菌细胞额外地编码能够水解细菌细胞的细胞质组分的酶,如WO03/006630中描述的。细菌菌影制备的相应方法包括下列额外步骤:
(a)任选地在其中所述酶基因不表达的条件下培养细菌细胞;
(b)将细菌细胞经历其中所述酶基因发生表达并且细菌细胞的细胞质组分被降解的条件。
编码水解酶的基因优选是核酸酶基因,特别地金黄色葡萄球菌(Staphylococcus aureus)核酸酶基因(WO03/006630)。
BG显示对广泛的测试细胞(包括噬菌体、树突细胞、肿瘤细胞、内皮细胞和上皮细胞)的活力和代谢活性没有细胞毒性和基因毒性影响。具有其完整表面结构的BG被专职APC(例如树突细胞和巨噬细胞)通过各种表面受体(例如补体受体和Toll样受体)高效识别和吞噬。此外,使用树突细胞(DC)作为最专职的抗原呈递细胞(专职APC)的模型的进一步研究显示,它们的吞噬细胞活性和BG的摄入依赖于用于产生BG的细菌株。
尽管裂解过程非常有效,但仍然可能存在潜在的污染(每10,000个BG约1个完整细菌细胞)。为了避免BG制剂中任何活细胞的存在(特别地在BG样品的冷冻干燥之前),优选在BG的最终收获之前将烷化剂(例如与核酸反应并引起核酸改变的β-丙内酯)添加至发酵***。符合人医学和兽医学应用标准的使用β-丙内酯进行最终灭活的生产方法公开于专利申请No.PCT/EP2009/000272中。
细菌菌影用作疫苗或佐剂的用途以及在其细胞包膜结构中具有异源蛋白质的重组细菌菌影的制备公开于专利申请No.PCT/EP98/04723中。
细菌菌影用作活性化合物的载体或靶向媒介物的用途公开于专利申请No.PCT/EP00/01906中。
根据本发明的组合物包含(i)抗原呈递细胞(APC),特别地专职抗原呈递细胞作为组分。在一个优选实施方案中,所述组合物包含单核细胞,最优选包含树突细胞(DC)。具体地,温育后并准备用于施用的本发明组合物包含载有成熟肿瘤相关抗原的DC,优选载有肿瘤相关抗原的DC是肿瘤相关抗原呈递树突细胞。
DC是最有效力的专职APC以及体内T细胞反应(包括MHC-限制的T细胞的敏化、T细胞依赖性抗体产生的发生以及免疫耐受性的诱导)的强效启动剂和调节剂。DC在周缘组织和次级淋巴组织中具有高吞噬细胞活性,并且通过几种机制(包括巨胞饮和受体介导的胞吞作用)捕获抗原(Ag)。DC的主要作用与外来抗源提供的潜在危险信号的识别、它们的内化、加工以及在MHC I类与II类分子的复合物内的呈递相关。在正常生理状况中的大多数情况下,DC以其特征在于高吞噬能力、共刺激分子和Ag呈递分子低表达和低细胞因子产生的未成熟状态存在。通过甘露糖受体(糖基化Ag的摄取)和Fc-受体(免疫球蛋白的摄取)进行的可溶性抗原的吞噬强有力地增强抗原呈递的效率。此外,与通过巨胞饮内化的可溶性Ag相比,Ag与抗体的复合物在通过Fc-受体内化后,DC能够以低于1/100的浓度呈递Ag。有效的T细胞刺激与DC成熟严格关联,其中DC成熟影响细胞因子的产生、共刺激分子的表达和肽-MHC复合物的呈递。通过内体-溶酶体途径进行的细胞外抗原的胞吞以及其加工通常导致MHC II类分子内的抗原片段的呈递。然而,通过Fc-受体介导的细胞外抗原的胞吞作用允许MHC I类和II类限制的抗原呈递并且诱导DC成熟。在MHC I类分子情形下细胞外抗原的呈递称为交叉呈递或交叉致敏。通过MHC分子进行的抗原的高效呈递与共刺激分子的表达和细胞因子分泌一起导致各种类型的T细胞(例如Th1、Th2、Treg或Th17)的刺激。对于活化Th1淋巴细胞及其增殖而言,成熟DC产生充足的IL-12是非常重要的。朝向Th1型T细胞免疫反应的极化被视为诱导有效的抗肿瘤免疫反应(从而导致肿瘤细胞的识别和消除)所必需的最重要因素之一。
BG显示极好的被专职APC(包括DC)识别和内化的能力。载有DNA的BG比裸露DNA更加高效地刺激小鼠的体液和细胞Ag-特异性免疫反应。在利用载有DNA的BG接种的动物中观察到了响应于由包含免疫显性MHC I类表位的肽脉冲的APC的再刺激而有产生IFN-γ的Ag-特异性CD8+T细胞的增加。此外,BG增强MHC I类分子和共刺激分子在DC上的表达。通过BG递送至DC的肿瘤相关Ag交叉提呈可活化CD4+和CD8+T细胞,并且刺激免疫***来增强抗由肿瘤表达的肿瘤相关Ag的免疫反应。
细菌脂多糖(LPS)增强DC的成熟,影响DC的内体酸化,并且还改善Ag的交叉提呈。BG的内膜和外膜结构(包括LPS)在蛋白E介导的革兰氏阴性细菌裂解后保持完整,因此,除了高负载能力以外,BG还在表面上“携带”用于刺激通过DC进行的交叉提呈的LPS-高效分子。
因此,本发明的组合物中APC与BG之间的相互作用导致APC的刺激、活化和从而成熟。
根据本发明的组合物还包含至少一种肿瘤相关抗原(组分(iii))。肿瘤相关抗原(TAA)可以例如由肿瘤细胞裂解物来提供。优选地,提供了自体肿瘤细胞裂解物,即来自来源于待治疗患者的肿瘤的裂解物。然而,还可能使用来自肿瘤细胞系的肿瘤相关抗原。
优选地,使用与待治疗的肿瘤相同类型的肿瘤的肿瘤细胞系。优选地,根据本发明的组合物包含至少两种不同的肿瘤细胞裂解物。
作为另外的选择,TAA可由BG携带。在一个实施方案中,TAA装载在BG上。在另一个实施方案中,携带TAA的BG是具有重组TAA(蛋白)的BG。这样的携带重组TAA的BG来源于重组表达TAA的细菌。
因此,本发明涉及其中组分(ii)和(iii)偶联在一起的组合物,其中例如以携带重组TAA(蛋白)的BG的形式偶联。
肿瘤或癌细胞优选来自选自如下的癌症:纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、成骨性肉瘤、脊索瘤、血管肉瘤、卡波西氏肉瘤、内皮肉瘤、***肉瘤、***内皮肉瘤、滑膜瘤、间皮瘤、Ewing瘤、平滑肌肉瘤、横纹肌肉瘤、横纹肌肉瘤、结肠直肠癌、结肠直肠腺癌、胰腺癌、乳腺癌、卵巢癌、***癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、***状癌、***状腺癌、囊腺癌、髓样癌、支气管原癌、肾细胞癌、肝细胞瘤、胆管癌、毛膜癌、***瘤、胚胎性癌、肾母细胞瘤、***、睾丸肿瘤、肺癌、小细胞肺癌、膀胱癌、上皮癌、神经胶质瘤、胶质母细胞瘤、星细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突神经胶质瘤、脑膜瘤、神经细胞瘤、视网膜母细胞瘤、骨髓瘤、淋巴瘤、黑素瘤和白血病。
在一个实施方案中,组合物包含T98G肿瘤细胞裂解物。
更优选地,组合物包含自体肿瘤细胞裂解物。
本发明的组合物可包含以热休克、化学处理的和/或杀死的形式存在的肿瘤细胞。
本发明的组合物还任选地包含细胞因子,特别地GM-CSF。此外,其任选地包含α-干扰素。
本发明特别地涉及及由单核细胞衍生的树突细胞(DC)、至少一种肿瘤细胞裂解物(优选至少两种来自相同肿瘤类型的不同肿瘤细胞裂解物)、细菌菌影(BG)和任选地重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和干扰素α(IFN-α)组成的抗肿瘤疫苗。
根据本发明的组合物中的抗原呈递细胞通过与肿瘤相关抗原(TAA)和细菌菌影(BG)一起温育来活化和/或成熟。具体地,一种或多种抗原呈递细胞被活化,所述抗原呈递细胞呈递一种或多种癌症抗原并且通过在所述一种或多种癌细胞存在的情况下(优选在一种或多种肿瘤或癌细胞裂解物存在的情况下),在细菌菌影存在的情况下温育所述一种或多种抗原呈递细胞来诱导T细胞活化。
本发明的一个有利方面是可在从自体患者肿瘤细胞和/或从相同组织学肿瘤类型制备的肿瘤细胞系获得的肿瘤裂解物存在的情况下温育DC。因此,可针对待治疗的各自癌症类型训练DC。自体肿瘤细胞的使用使其它疾病的感染或转移的风险降至最低。
另一个有利方面是在由两种或更多种获自相同肿瘤类型的不同细胞系制备的肿瘤裂解物存在的情况下温育DC产生的。因此,由DC呈递的抗原的范围可得到增加。
BG的完整表面免疫刺激性结构(任选地与IFN-α和/或GM-CSF一起)刺激DC的完全成熟并引发IL-12的产生。GM-CSF是通常用于产生成熟DC的细胞因子。
单核细胞可通过抽血或白细胞去除术从患者的外周血获得,这保证了充足量的细胞。
与一种或多种肿瘤裂解物的温育提供了几种肿瘤相关抗原(TAA)的存在并且导致更广的TAA特异性免疫反应的刺激。
肿瘤裂解物可从在患者的手术过程中获得的肿瘤组织的自体样品或从由相同肿瘤类型产生的肿瘤细胞系制备而来。在手术之前对组织供体进行性传播疾病(STD)例如HIV、HCV、HBV、梅毒筛查,仅阴性患者被视为组织供体。
可将基于DC、肿瘤裂解物和BG的抗肿瘤疫苗用作用于具有肿瘤,特别地具有胶质母细胞瘤,肾癌、卵巢癌、***癌、膀胱癌、结直肠癌、结直肠腺癌和黑素瘤的患者的治疗。优选地,从各自肿瘤类型和/或肿瘤细胞系制备肿瘤裂解物。
根据本发明的组合物优选包含空细菌菌影作为组分(iii)。然而,还可能使用特别地载有免疫***的激活剂或沉默子的细菌菌影。细菌菌影可特别地载有药物试剂和/或DNA。
组合物可任选地包含细菌脂多糖(LPS)。
已发现本发明组合物中包含的并且与TAA以及BG温育的APC,特别地树突细胞产生特定细胞因子,具体地IL-12、IL-23和/或IL-1β。
当给患者施用成熟的APC(特别地DC)时,免疫反应被诱导。特别地,已发现,本发明的组合物诱导Th17反应,该反应在施用未曾与细菌菌影温育过的抗原呈递细胞的组合物时未被观察到。
根据本发明的组合物优选地以每个抗原呈递细胞计,具体地以每个树突细胞计至少一个细菌菌影,更优选至少5个细菌菌影,更优选至少10个或至少100个细菌菌影。组合物可包含以每个树突细胞计达到10,000个细菌菌影,优选达到1,000个细菌菌影,更优选达到500个细菌菌影。
本发明还涉及包含本文中描述的组合物的疫苗。通过施用这样的疫苗,诱发抗癌细胞的免疫反应。因此,本发明还涉及用于肿瘤免疫疗法的疫苗。当施用时,根据本发明的组合物以及具体地其中包含的抗原呈递细胞诱发增强的肿瘤抗原识别,从而自体T细胞进行的肿瘤特异性杀伤。
对于施用,疫苗优选包含每剂量约100万至1000万个细胞,具体地400万至600万个细胞。优选地,施用5至15,具体地6至10个剂量。可以数周的时间进行施用。优选每周一次施用前2至3个剂量,随后每月一次施用剩余的剂量。
对于贮存,可将组合物冷冻或冷冻干燥。优选冷冻组合物。
可以以任何适当的方式(例如全身性、皮下、结节内、皮内或瘤内)进行施用。优选地,通过皮下、结节内、皮内或瘤内施用组合物。
对于施用,细菌菌影可包含用于活化或沉默免疫***的活性剂。为了使APC成熟,细菌菌影可包含额外的成分,例如肿瘤裂解物、肿瘤肽、肿瘤抗原或细胞因子。与细菌菌影和肿瘤相关抗原一起温育抗原呈递细胞优选进行10min至12h,优选15min至6h,更优选3h至5h的时间范围。
在一个特别优选实施方案中,将树突细胞培养在针对DC的产生、临床离体使用最优化的、遵照相关GMP-指导方针标准化、制造、测试和释放的商购可得的DC培养基GMP无血清培养基中。
优选通过在+37℃、5%CO2潮湿培养箱中于补充有DNA酶的培养基中温育单核细胞(特别地获自患者外周血的DC)2h,随后在补充有重组人GM-CSF和IFN-α的培养基中温育3天(以获得未成熟DC群体)来制备上述刺激剂和DC组合的抗肿瘤疫苗。将至少一种肿瘤裂解物,优选至少两种从相同肿瘤类型的至少两种不同细胞系制备的肿瘤裂解物与BG混合在一起,涡旋并在轻轻摇动的条件下在室温(RT)温育1h。随后,将重组人GM-CSF和IFN-α与BG与肿瘤裂解物的混合物混合,将其添加至未成熟DC中,于5%CO2潮湿培养箱中+37℃下温育。
在10min至12h的温育后,更优选4h的温育后,通过将来自DC的培养基轻轻地收集至无菌50ml管内并以700RPM/5分钟/RT离心沉淀来小心地除去未内化的肿瘤裂解物和BG。同时,将补充有重组人GM-CSF和IFN-α的新鲜培养基添加至剩余细胞。离心后,迅速并小心地除去上清液,将沉淀重悬浮于补充有重组人GM-CSF和IFN-α的培养基中,并将细胞返回至培养瓶。将DC于5%CO2潮湿培养箱中在+37℃温育另外6h,随后深度冷冻疫苗。
本发明通过附图和下列实施例来进一步描述。
图1.细胞表面标志物在DC上的表达。通过在补充有IFN-α(3000IU/mL)和rhGM-CSF(1000IU/mL)的DC培养基中温育获自正常健康供体的外周血的单核细胞3天来制备未成熟DC。通过在有IFN-α和rhGM-CSF存在下用肿瘤裂解物和来自大肠杆菌Nissle1917的BG(10和100个BG/1DC)短暂(4h)刺激未成熟DC后48h,利用多色流式细胞术(multicolor flow cytometry)分析DC的成熟标志物。将与IFN-α和rhGM-CSF以及补充有脂多糖(LPS)(200ng/mL)或无额外成熟刺激物的肿瘤裂解物温育过的未成熟DC用作对照。Y轴代表表达特异性分化抗原的细胞的百分比。数据代表使用获自不同供体的细胞进行的4个独立实验的平均值±SD。
CD14是树突细胞成熟的指示剂。单核细胞显示CD14表达,而成熟树突细胞不显示或几乎不显示CD14表达。
CCR7是细胞至***的诱引剂的标志物。如可从图1看到的,对于利用细菌菌影温育过的树突细胞,标志物CCR7的表达增加,显示了它们的运动。
CD83是成熟树突细胞的主要标志物。
CD80、CD86、CD1a、CD11c和HLA-DR是树突细胞的成熟标志物。
图2.利用肿瘤裂解物和LPS温育后或利用BG短暂刺激后的DC的迁移能力。在IFN-α、rhGM-CSF和补充有来自大肠杆菌Nissle1917的BG(10和100个BG/1DC)、LPS(200ng/mL)或无额外成熟刺激物的肿瘤裂解物存在的情况下响应CCL21(通过结合至细胞表面趋化因子受体CCR7引发其作用的趋化因子)而成熟的不同DC群体的趋化作用测定为迁移进入包含不同浓度的CCL21的转孔***(transwell system)的下层部分的细胞的数目,通过流式细胞仪计数。每一个条块代表使用获自不同正常健康供体的细胞进行的4个独立实验的平均迁移细胞数目±SD。
图3.在肿瘤细胞裂解物和LPS存在的情况下或利用BG的短时间温育下成熟的DC的细胞因子模式。在于24h和48h温育后在测量释放进入上清液的细胞因子之前,在纯LPS(200ng/mL)或来自大肠杆菌Nissle1917的BG(10和100个BG/1DC)存在的情况下利用IFN-α、rhGM-CSF和肿瘤裂解物短时间(4h)刺激未成熟DC。在无另外的刺激物存在的情况下温育的细胞用作阴性对照。使用FACSArray Bioanalyzer测量从DC释放的细胞因子的水平。数据代表使用获自不同正常健康供体的细胞进行的4个独立实验的平均值±SD。P值<0.05被认为是显著的,并且利用星号来指示(*,P<0.05;**,P<0.01;***,P<0.001)。
IL-12和IFN-γ是T淋巴细胞分化成Th1型淋巴细胞的主要活化标志物。
IL-23(生长和稳定化因子)和IL-6(分化因子)是参与Th17淋巴细胞的发育的细胞因子。图3显示T辅助细胞被本发明的组合物诱导。
IL-1β和TNF-α是促炎标志物。
IL-10是抗炎细胞因子。
IL-2是T细胞生长因子。
图4.分析的DC的同种异体(A)和自体(B)免疫刺激能力。相较于利用IFN-α、rhGM-CSF和补充有纯LPS或无额外成熟刺激物的肿瘤裂解物而成熟的DC而言,利用IFN-α、rhGM-CSF、肿瘤裂解物和BG短时间(4h)温育未成熟DC显示增强了DC刺激自体T细胞增殖的能力。在于荧光标记的(CFSE-标记的)同种异体或自体T细胞以1:10的DC:T细胞比率存在的情况下温育6天后测定分析的DC群体的自体和同种异体免疫刺激能力。在用一小组单克隆抗体(抗-CD3、抗-CD4和抗-CD8)温育后对刺激的细胞进行染色,通过多色流式细胞术测定自体和同种异体T细胞的增殖。将数值计算为从用不同DC群体刺激后增殖的细胞的百分比扣除自发地增殖的T细胞的百分比。利用植物凝集素(PHA)(5μg/ml)温育的T细胞用作阳性对照。数据代表使用获自不同供体的细胞进行的4个独立实验的平均值±SD。P值<0.05被认为是显著的,并且用星号标示(*,P<0.05;**,P<0.01;***,P<0.001)。
图5.在用BG短时间刺激后由载有肿瘤裂解物的DC诱导的同种异体(A)和自体(B)T细胞的肿瘤细胞识别和细胞毒性作用。在利用纯LPS(200ng/mL)预刺激的、利用来自大肠杆菌Nissle1917的BG(10和100个BG/1DC)或不利用额外成熟刺激物短时间温育(4h)的载有肿瘤细胞裂解物(T98G细胞)的DC存在的情况下将同种异体或自体T细胞温育6天。随后,以10:1的效应子:靶比率将经刺激的T细胞添加至新鲜荧光标记的(CFSE-标记的)T98G肿瘤细胞。在相互共温育后24h,利用流式细胞术测定肿瘤细胞的特异性裂解。利用10%Et-OH温育后的肿瘤细胞的裂解用作阳性对照(100%)。在预刺激过的效应细胞不存在的情况下温育的肿瘤细胞用作自发细胞死亡的阴性对照。数据代表使用获自不同供体的细胞进行的4个实验的平均值±SD。P值<0.05被认为是显著的,并且用星号标示(*,P<0.05;**,P<0.01;***,P<0.001)。
具体地,相较于单独的DC或DC+LPS而言,对于DC+BG,T98G肿瘤细胞处理的结果显示相当高的增强。
在图1至5中显示的所有实验中,包括人树突细胞。
实施例
实施例1.细菌菌影.
按照专利申请No.PCT/EP2009/000272制备细菌菌影。
实施例2.肿瘤裂解物制备
将获自癌症患者或组织培养板(瓶)的肿瘤组织贮存在装有NaCl(0.9%的注射用水“Fresenius”中的氯化钠)的管中。将具有肿瘤组织或细胞的管于+4℃贮存并在手术后处理达3天。将管进行辐照(120Gy),随后处理组织。必须对供体(患者)进行性传播疾病(STD)例如HIV、HCV、HBV、梅毒筛查,排除上述任何疾病检测阳性的患者。将获自患者的肿瘤组织转移至装有5-10ml HBSS(Hanks′平衡盐溶液;Lonza,No.:10-547F或10-527F;按照cGMP规则生产)的无菌Petri培养皿。通过解剖刀和镊子除去坏死组织和***。将其余肿瘤组织切成约5mm的小块并通过无菌注射器柱塞进行研磨。通过将悬浮液经过连接至无菌注射器的20G(0.9mm)针数次、直至获得匀浆悬浮物来进一步对获得的细胞悬浮物进行匀浆。随后将细胞悬浮物经过尼龙滤过器(100μm)进行过滤,将其收集在无菌50ml管中。用剩余HBSS洗涤Petri培养皿,通过尼龙滤过器过滤,随后与过滤的细胞混合。将细胞悬浮物以1600RPM在+4℃离心沉淀7min。迅速而小心地倾倒上清液,将沉淀重悬浮于2ml的培养基中。将单细胞悬浮物等量地分配入微量管。将微量管置于液N2或干冰与甲醇的混合物中进行约3分钟。随后,将冷冻的细胞在室温(RT)解冻15-30分钟直至细胞沉淀变得完全融化(不应当将细胞保持在RT太久以免肿瘤细胞蛋白降解)。应当重复冷冻-解冻过程5次。随后在超声波浴中对细胞裂解物超声5分钟。将细胞碎片与细胞裂解物一起收集在一个管中,以10000-15000RPM离心沉淀10min,随后使用细针分离出沉淀来迅速小心地收集上清液(细胞裂解物)。不应当过滤细胞裂解物,必须在无菌条件下进行所有步骤。将未过滤细胞裂解物等分,于-80℃下贮存直至进一步使用。用10x DPBS(Dulbecco′s磷酸缓冲盐溶液,10xPBS,Lonza,No.:17-515F)稀释细胞裂解物。使用分光光度计来测定蛋白质浓度。
实施例3.用于抗肿瘤疫苗制备的单核细胞来源的树突细胞的培养
通过淘洗(Elutra Cell Separation System,CaridianBCT EuropeNV/SA)或磁性分离(CliniMACS,Miltenyi Biotec GmbH)分离获自患者外周血的单核细胞。将GM-CSF溶解在培养基中以获得1x105IU/ml的终浓度并等分入微量管(每管700μl),并于-80℃贮存。将IFN-α(Roche,ROFERON-A12x106IU/ml)等分进入微量管(每管17.5μl)并于+2℃至+8℃贮存。在无菌条件下将低温保存的冷冻培养基CryoStor CS2或CryoStor CS5等分进入微量管(每管1.5ml)。将分离的单核细胞重悬浮于培养基中,并添加至培养瓶中,约167x106个单核细胞于70ml的培养基中。对于一个培养瓶需要约150μg的肿瘤裂解物,或来自相当于未成熟DC(单核细胞)数目3倍的肿瘤细胞数的细胞的肿瘤裂解物。以1500RM在RT离心来自培养瓶的单核细胞悬浮物10min。在倾倒上清液后,将细胞沉淀重悬浮于小体积的培养基中,利用培养基补充达到35ml。将细胞悬浮物转移至培养瓶中。将35ml培养基中的GM-CSF(700μl/7x104IU)和IFN-α(17.5μl/2.1x105IU)添加至单核细胞悬浮液中。通过从一侧至另一侧轻轻地移动培养瓶来细心地混合培养瓶的内容物。将细胞在5%CO2潮湿培养箱中在+37℃温育3天。
收集制备的未成熟DC的细胞悬浮物,分配入3个无菌50ml管。将新鲜培养基(5ml)同时添加至每一个培养瓶中以避免附着在培养瓶表面上的细胞死亡。将细胞悬浮物以1500RPM在RT下离心10分钟。快速而小心地倾倒上清液,将细胞沉淀重悬浮于2ml的培养基中,将来自培养瓶的细胞转移至1个管中。用4ml的培养基轻轻地漂洗用于离心细胞的每一个管,将内容物转移至具有收集细胞的管。将获自肿瘤组织或肿瘤细胞系的肿瘤裂解物(5ml)(肿瘤细胞:DC的比率=3:1;501x106:167x106)与从大肠杆菌Nissle1917制备的167x108个细菌菌影(BG:DC的比率=100:1)混合,充分涡旋,在RT在轻轻摇动的条件下温育60min。将BG与补充有GM-CSF(25μl/2.5x104IU)和IFN-α(6.25μl/7.5x104IU)的肿瘤细胞裂解物的混合物添加至DC悬浮物。随后,将细胞悬浮物与所有试剂转移至培养瓶。将包含细胞悬浮物的管用5ml的培养基进行漂洗,随后将培养基转移至培养瓶,在从一侧至另一侧地轻轻摇动的情况下小心地混合。将细胞在5%CO2潮湿的培养箱中于+37℃温育4小时以允许BG和肿瘤裂解物内化,并起始成熟过程。在4h的温育后,将细胞转移至50ml管,用培养基轻轻地漂洗培养瓶。将用于培养瓶漂洗的培养基转移至具有细胞悬浮物的管中。将新鲜培养基(5ml)同时添加至培养瓶中以避免附着至培养瓶表面的细胞死亡。将细胞悬浮物以1500RPM在RT离心沉淀10分钟。迅速而小心地倾倒上清液,将细胞沉淀重悬浮于20ml补充有GM-CSF(25μl/2.5x104IU)和IFN-α(6.25μl/7.5x104IU)的新鲜培养基中。将细胞悬浮物转移至小心并且轻轻地从一侧至另一侧摇动的原始培养瓶内,将细胞在5%CO2潮湿培养箱中于+37℃温育另外6h。
实施例4.抗肿瘤疫苗制剂的贮存.
将具有在实施例3中获得的刺激DC的培养瓶充分摇动以释放附着至培养瓶壁的细胞。将细胞悬浮物完全转移至标记的50ml管中。如果可能的话,用两个额外体积的用于肿瘤裂解物制备的相同HBSS(10ml)漂洗培养瓶,并且将整个体积的HBSS转移至具有细胞悬浮物的管中。原始培养瓶装有5ml HBSS,将其放回培养箱中。用HBSS将细胞悬浮物的体积补足到50ml,通过管的颠倒轻轻混合。将细胞悬浮物以1500RPM在+4℃离心沉淀10分钟。迅速而小心地倾倒上清液,首先将细胞悬浮物重悬浮于5ml HBSS中,随后添加另外的45ml HBSS,通过管的颠倒充分混合。使用Bürkner计数室将10μl细胞悬浮物用于测定细胞数目。如果悬浮物中细胞的浓度低于1.4x106/ml,应当用accutase从培养瓶释放剩余的细胞,另外地将各自包含5x106个细胞的细胞等分冷冻以待将来给患者施用。必须制备至少6个等分以给患者施用,一个等分用于质量控制,一个等分用于免疫监控,一个等分用于测试支原体以及3个等分用于使用(arbitrage)。将细胞悬浮物以1500RPM在+4℃离心沉淀10分钟。将冷冻管转移至预冷的MiniCooler中。迅速而小心地倾倒上清液,将细胞沉淀重悬浮于冷藏保存的冷冻培养基CryoStor CS2中并且转移至冷冻管。在等分抗肿瘤疫苗制剂后立即将所有冷冻管置于-80℃异丙醇盒中。在于-80℃进行24h后,将所有冷冻疫苗制剂转移至专门针对抗肿瘤疫苗制剂目的设置的装有液氮的Dewar容器内。
表1-在给患者施用前抗肿瘤疫苗制剂的可接受性的标准
*在给患者施用抗肿瘤疫苗制剂之前,至少3至5个表型标志物应当满足通过质量控制的标准。
**在给患者施用抗肿瘤疫苗制剂之前,同种异体MLR测试中3个检查的DC:PBMC比率中至少2个应当满足通过质量控制的标准。
Claims (17)
1.一种组合物,其中包含
(i)抗原呈递细胞,
(ii)肿瘤相关抗原,和
(iii)细菌菌影。
2.权利要求1的组合物,其用作用于肿瘤免疫疗法的疫苗。
3.权利要求1或2的组合物,其中所述抗原呈递细胞包含单核细胞。
4.前述权利要求中任一项的组合物,其中所述抗原呈递细胞包括树突细胞。
5.前述权利要求中任一项的组合物,其包含一种或多种肿瘤裂解物。
6.前述权利要求中任一项的组合物,其包含获自包含编码裂解蛋白质的基因的细菌细胞的细菌菌影。
7.前述权利要求中任一项的组合物,其包含已用β-丙内酯处理的细菌菌影。
8.前述权利要求中任一项的组合物,其包含来自革兰氏阴性细菌,具体地来自大肠杆菌,更优选来自大肠杆菌Nissle1917的细菌菌影。
9.前述权利要求中任一项的组合物,其包含载有免疫***的激活物或沉默子、药物试剂和/或DNA的细菌菌影。
10.前述权利要求中任一项的组合物,其包含携带有重组肿瘤相关抗原的细菌菌影。
11.权利要求10的组合物,其中所述细菌菌影装载有肿瘤相关抗原。
12.权利要求10的组合物,其中所述细菌菌影来源于重组表达肿瘤相关抗原的细菌。
13.前述权利要求中任一项的组合物,其中每个抗原呈递细胞包含1至10,000个,具体地10至1,000个细菌菌影。
14.前述权利要求中任一项的组合物,其中每剂量包含100万至1000万个抗原呈递细胞。
15.权利要求2的组合物,其包含诱导通过T细胞进行的肿瘤抗原识别和肿瘤特异性杀伤的抗原呈递细胞。
16.权利要求2的组合物,其包含自体抗原呈递细胞。
17.一种治疗性疫苗,其包含前述权利要求中任一项的组合物。
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US20160129097A1 (en) * | 2014-11-06 | 2016-05-12 | Jeremy Delk | Method of processing a veterinary tumor vaccine and a veterinary tumor vaccine processing kit |
CN105126094A (zh) * | 2015-08-13 | 2015-12-09 | 安徽农业大学 | 一种拟态弧菌菌影疫苗及其制备方法和应用 |
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