CN116421734A - 适用于溶瘤病毒的冻干保护剂及其应用 - Google Patents
适用于溶瘤病毒的冻干保护剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种适用于溶瘤病毒的冻干保护剂,其为具有功能成分的溶液,以工作浓度下的质量百分数计,所述功能成分包括:蔗糖3%‑10%、明胶0.3%‑1.0%、人血清白蛋白(HSA)0.1%‑1.0%和谷氨酸钠0.1%‑1.0%。相比传统的溶瘤病毒液体制剂,其冻干制剂更容易保存,稳定性好,尤其是冻干后的滴度和肿瘤杀伤能力表现良好。
Description
技术领域
本发明涉及溶瘤病毒技术领域,具体涉及一种适用于溶瘤病毒的冻干保护剂及其应用。
背景技术
随着生命科研技术的提高,肿瘤癌症治疗出现了不少新型热门疗法,除了单/双抗、ADC(抗体偶联药物)、PD-1/L1之外,细胞疗法、外泌体、溶瘤病毒等也都成为了该领域的治疗手段。其中,溶瘤病毒(Oncolytic viruses,OVs)因其能够特异性地在肿瘤细胞内复制并造成肿瘤细胞裂解而不影响正常细胞,受到越来越多科研和产业的重视。溶瘤病毒是一类天然的或经基因工程改造的,可选择性地在肿瘤组织内复制,进而感染杀伤肿瘤细胞或导致肿瘤细胞裂解,但对正常组织无杀伤作用的病毒。根据其是否进行过基因改造,主要可以分为两类:一类是野生型病毒株和天然的弱毒病毒株,比如呼肠孤病毒,新城疫病毒等;另一类是经过基因改造只能在肿瘤细胞内进行增殖的病毒,主要有腺病毒、单纯疱疹病毒、牛痘病毒以及麻疹病毒等。
溶瘤病毒疗法(Oncolytic virotherapy)是一种新型的肿瘤靶向疗法,它是利用肿瘤选择性、多杀伤途径的溶瘤病毒作为主要成分的抗肿瘤制剂,其能够直接杀伤肿瘤细胞达到溶瘤效果,并在肿瘤细胞裂解的过程中释放肿瘤特异性抗原,激活机体抗肿瘤免疫应答。溶瘤病毒可利用多种作用机制杀死癌细胞,所述机制包括细胞溶解、细胞凋亡、抗血管生成和细胞坏死。病毒感染肿瘤细胞然后开始复制。病毒继续复制直到最后“溶解”(裂解)宿主细胞的膜,肿瘤细胞不再能含有病毒。肿瘤细胞被摧毁并且新产生的病毒扩散到邻近的癌细胞继续该循环。值得注意的是所有溶瘤病毒仅在癌细胞中复制,其通过正常组织时不导致损害,这很重要。因此,一旦根除所有肿瘤细胞,溶瘤病毒不再具有复制的能力并且免疫***能够将其从体内清除。
由于冻干制剂可以有效的保护产品,使其保持原有的生物学结构和物理性质,所以已经广泛应用到疫苗类生物制品中。然而,目前,很少有适用于溶瘤病毒的冻干保护剂的研究,大多数的冻干保护剂还是适用于传统的减毒或灭活疫苗,经传统冻干保护剂冻干后的溶瘤病毒的毒力变差,与冻干前相比滴度变差,并且冻干后的热稳定性变差,体现在冻干后的溶瘤病毒在用于体内治疗时溶瘤病毒在体内长时间存在时的滴度逐渐变差,进而影响溶瘤病毒对肿瘤的杀伤效果。因此目前市场上的溶瘤病毒制品均为液体制剂,相比冻干制剂,液体制剂缺点很多,如:保存和运输的成本高,通常需要-80℃超低温保存。风险也高,如果冷库或者冰箱故障,温度上升导致制品融化,滴度会下降一半左右,使制品报废。
发明内容
基于此,有必要提供一种适用于溶瘤病毒的冻干保护剂及其应用。
本发明的第一目的在于提供一种适用于溶瘤病毒的冻干保护剂,其为具有功能成分的溶液,以工作浓度下的质量百分数计,所述功能成分包括:
蔗糖3%-10%、明胶0.3%-1.0%、人血清白蛋白(HSA)0.1%-1.0%和谷氨酸钠0.1%-1.0%
本发明的第二目的在于提供所述的冻干保护剂的制备方法,包括:
将灭菌后的各组分溶于溶剂中,混匀。
本发明的第三目的在于提供所述的溶瘤病毒冻干粉的制备方法,包括:
将溶瘤病毒与所述的冻干保护剂混合后再冻干。
本发明的第四目的在于提供所述的溶瘤病毒冻干粉的制备方法制备得到的溶瘤病毒冻干粉。
本发明的第五目的在于提供所述的溶瘤病毒冻干粉在制备用于杀伤肿瘤的药物中的应用。
本发明提出了一种用于制备溶瘤病毒的冻干粉制备的冻干保护剂,相比传统的溶瘤病毒液体制剂,其冻干制剂更容易保存,稳定性好。该冻干保护剂的功能成分为蔗糖、明胶、HSA、谷氨酸钠,或者还含有精氨酸、尿素、甘油。上述几种成分在合适的配比下相互配合,能够使得溶瘤病毒在冻干过程中其活性损失较小,冻干前后的滴度损减不大,并且重要的是,溶瘤病毒在使用本申请的冻干保护剂,冻干前后对肿瘤细胞的杀伤能力相差在+40%~-20%以内(+40%表示冻干后肿瘤杀伤能力提升了40%;-20%表示冻干后肿瘤杀伤能力降低了20%)。。而且,冻干后的制剂能够在机体温度下(37℃)长时间保持稳定性,有利于作为肿瘤杀伤药物应用于机体内,提高肿瘤杀伤的时间。
附图说明
图1为本发明实施例2的不同样品的细胞因子IL-12的表达图;
图2为本发明实施例2的不同样品的细胞因子IL15的表达图;
图3为本发明实施例2的不同样品的hIgG4的表达图;
图4为本发明实施例3的使用a3冻干保护剂进行批次1样品冻干前对肝癌细胞Hep3B的杀伤能力图;
图5为本发明实施例3的使用a3冻干保护剂进行批次2样品冻干前对肝癌细胞Hep3B的杀伤能力图;
图6为本发明实施例3的使用a3冻干保护剂进行批次1样品冻干后对肝癌细胞Hep3B的杀伤能力图;
图7为本发明实施例3的使用a3冻干保护剂进行批次2样品冻干后对肝癌细胞Hep3B的杀伤能力图;
图8为本发明实施例2的使用a3冻干保护剂的两个批次的样品冻干前后,以及加速稳定性试验中的滴度变化。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本文中所使用的术语“含有”、“包含”和“包括”是同义词,其是包容性或开放式的,不排除额外的、未被引述的成员、元素或方法步骤。
用端点表示的数值范围包括该范围内所包含的所有数值及分数,以及所引述的端点。
当描述一个可测量的值,例如参数,量,时间期限等时,本文中所使用的术语“约”意欲涵盖与指定值相差+/-20%或更少、优选+/-10%或更少、更优选+/-5%或更少、更优选+/-1%或更少,更优选+/-0.1%或更少的变化,此类变化适宜在所披露的发明中使用。
第一方面,本发明实施例提供一种适用于溶瘤病毒的冻干保护剂,其为具有功能成分的溶液,以工作浓度下的质量百分数计,所述功能成分包括:
蔗糖约3%-约10%、明胶约0.3%-约1.0%、HSA约0.1%-约1.0%和谷氨酸钠约0.1%-约1.0%。
在本申请中,所述冻干保护剂还可包括精氨酸,以工作浓度下的质量百分数计,所述精氨酸为约0.1%-约1.0%。
在本申请中,所述冻干保护剂还可包括甘油,以工作浓度下的质量百分数计,所述甘油为约0.01%-约0.15%。
在本申请中,所述冻干保护剂还可包括尿素,以工作浓度下的质量百分数计,所述尿素为约0.1%-约1.0%。
本发明实施例的用于制备溶瘤病毒的冻干粉制备的冻干保护剂,相比传统的溶瘤病毒液体制剂,其冻干制剂更容易保存,稳定性好。该冻干保护剂的功能成分为蔗糖、明胶、HSA、谷氨酸钠,或者还含有精氨酸、尿素、甘油。上述几种成分在合适的配比下相互配合,能够使得溶瘤病毒在冻干过程中其活性损失较小,冻干前后的滴度损减不大,并且重要的是,溶瘤病毒在使用本申请的冻干保护剂,冻干前后对肿瘤细胞的杀伤能力相差在+40%~-20%以内(+40%表示冻干后肿瘤杀伤能力提升了40%;-20%表示冻干后肿瘤杀伤能力降低了20%)。而且,冻干后的制剂能够在机体温度下(37℃)长时间保持稳定性,有利于作为肿瘤杀伤药物应用于机体内,提高肿瘤杀伤的时间。
冻干制剂与液体制剂最大区别在于:冻干制剂可以去掉绝大部分的水分,并且储存基本是无氧条件,制品不易氧化,制品的稳定性更好,保质期更长。冻干制剂对保存条件要求降低,运输和保存的成本降低,风险也降低。用药的容错率降低,不用担心用药前病人突发状况,而延后用药或不用药。
在本发明中,术语“冻干”和“冷冻干燥”在本文中可互换使用和是指通过首先冷冻和然后减少周围的压力以允许物质中的冷冻的水升华而脱水的物质。物料可先在冷冻装置内冷冻,再进行干燥。但也可直接在干燥室内经迅速抽成真空而冷冻。
本发明的冻干保护剂进行溶瘤病毒冻干制剂的制备中,冻干方法可以为常规的病毒或减毒病毒或疫苗的冻干方法。在一些具体实施方式中,例如可以包括步骤:预冻阶段:最低温度≤-40℃,达到最低温度后维持2~3小时;升华干燥阶段:最终温度-15~-10℃,达到最终温度的时间为8~14小时,真空压力控制在8~10Pa;解吸干燥阶段:最终温度34~37℃,真空压力控制在0.5~10Pa,运行15~22小时。
在本发明中,术语“工作浓度”用于限定所述冻干保护剂在处理微生物时主要活性组分的比例,所述冻干保护剂中活性组分的浓度可以为工作浓度,也可以为能够稀释为该浓度的母液(例如2、3、4、5、6、7、8、9、10、20、30、40、50倍浓缩的母液)。
在一些实施方式中,以工作浓度下的质量百分数计,所述功能成分由以下组分组成:
蔗糖约3%-10%、明胶约0.3%-1.0%、HSA约0.1%-1.0%、谷氨酸钠约0.1%-1.0%、尿素约0.1%-1.0%和精氨酸约0.1%-1.0%。
在另一些实施方式中,以工作浓度下的质量百分数计,所述功能成分由以下组分组成:蔗糖约3%-9%、明胶约0.3%-0.9%、HSA约0.1%-0.9%、谷氨酸钠约0.1%-0.9%、尿素约0.1%-0.9%和精氨酸约0.1%-0.9%。在一些实施方式中,所述溶液的溶剂为水,例如去离子水,无菌水。
具体的,蔗糖的含量还可以为4.5%、5.0%、5.5%、6.0%、6.5%、7.0%、7.5%、8.0%、8.5%或9.0%。明胶的含量还可以为0.45%、0.50%、0.55%、0.60%、0.65%、0.70%、0.75%、0.80%、0.85%、0.90%或0.95%。HSA的含量还可以为0.17%、0.18%、0.19%、0.20%、0.21%、0.22%、0.24%、0.26%、0.28%或0.30%。谷氨酸钠的含量还可以为0.07%、0.08%、0.09%、0.10%、0.11%、0.12%、0.13%、0.14%、0.16%、0.18%或0.20%。尿素的含量还可以为0.45%、0.50%、0.55%、0.60%、0.65%、0.70%、0.75%或0.80%。
具体的,精氨酸的含量还可以为0、0.01%、0.02%、0.03%、0.04%、0.05%、0.06%、0.07%、0.08%、0.09%、0.10%、0.11%、0.12%、0.14%、0.16%、0.18%或0.20%。
在本申请中,所述冻干保护剂可包含蔗糖3%-8%、明胶0.3%-1.0%、HSA0.1%-1.0%和谷氨酸钠0.1%-1.0%(以工作浓度下的质量百分数计)。在某些实施方式在,所述冻干保护剂还包含精氨酸0.1%-1.0%(以工作浓度下的质量百分数计)。
在本申请中,所述冻干保护剂可包含蔗糖4%-7%、明胶0.3%-0.8%、HSA0.2%-0.6%和谷氨酸钠0.1%-0.5%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含精氨酸、甘油和/或尿素。
在本申请中,所述冻干保护剂可包含蔗糖4%-7%、明胶0.3%-0.8%、HSA0.2%-0.6%和谷氨酸钠0.1%-0.5%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含精氨酸0.1%-0.5%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含尿素0.1%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含甘油0.1%(以工作浓度下的质量百分数计)。
在本申请中,所述冻干保护剂可包含蔗糖5%-6%、明胶0.5%-0.6%、HSA0.1%-0.2%和谷氨酸钠0.1%-0.3%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含精氨酸、甘油和/或尿素。
在本申请中,所述冻干保护剂可包含蔗糖5%-6%、明胶0.5%-0.6%、HSA0.1%-0.2%和谷氨酸钠0.1%-0.3%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含精氨酸0.1%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含尿素0.5%(以工作浓度下的质量百分数计)。在某些实施方式中,所述冻干保护剂还包含甘油0.05%(以工作浓度下的质量百分数计)。
在本申请中,所述冻干保护剂可包含蔗糖5%-6%、明胶0.5%-0.6%、HSA0.1%-0.2%、谷氨酸钠0.1%-0.3%、精氨酸0.1%-0.15%和尿素0.5%-0.6%(以工作浓度下的质量百分数计)。
在本申请中,所述冻干保护剂可包含蔗糖5%-6%、明胶0.5%-0.6%、HSA0.1%-0.2%、谷氨酸钠0.1%-0.3%、精氨酸0.1%-0.15%和甘油0.05%-0.06%(以工作浓度下的质量百分数计)。
在本申请中,所述冻干保护剂可包含蔗糖5%-6%、明胶0.5%-0.6%、HSA0.1%-0.2%、谷氨酸钠0.1%-0.3%和尿素0.5%-0.6%(以工作浓度下的质量百分数计)。
在本申请中,所述冻干保护剂可包含蔗糖5%-6%、明胶0.5%-0.6%、HSA0.1%-0.2%、谷氨酸钠0.1%-0.3%和甘油0.05%-0.06%(以工作浓度下的质量百分数计)。
任选地,冻干保护剂中还可以含有缓冲物质,本文使用的术语“缓冲物质”指水溶液或组合物,当酸或碱加入该溶液或组合物中时,所述水溶液或组合物抵抗pH中的变化。这种对pH变化的抗性是由于此类溶液的缓冲性质。因此,显示出缓冲活性的溶液或组合物被称为缓冲液或缓冲溶液。缓冲液一般不具有无限的维持溶液或组合物的pH的能力。相反,它们一般能够维持在特定范围内的pH,例如pH 7~pH 9。一般地,缓冲液能够维持在其pKa上和下一个对数内的pH(参见例如,Mohan,Buffers,A guide for the preparation anduseof buffers in biological systems,CALBIOCHEM,1999)。缓冲液和缓冲溶液一般由缓冲盐或优选非离子缓冲液组分如Tris和HEPES制备。可以在本发明的方法中使用的缓冲液优选选自磷酸盐缓冲液、磷酸盐缓冲盐水缓冲液(PBS)、2-氨基-2羟甲基-1,3-丙二醇(Tris)缓冲液、TRIS缓冲盐水溶液(TBS)、TRIS/EDTA(TE)、Earle's平衡盐溶液(EBSS)。
例如,本申请所述冻干保护剂可包含EBSS作为缓冲液。又例如,本申请所述冻干保护剂可包含Tris-HCl作为缓冲液。又例如,本申请所述冻干保护剂可包含Tris作为缓冲液。本申请的冻干保护剂可包含一种或多种(例如,两种)缓冲液。
在一些实施方式中,所述冻干保护剂的工作酸碱度为pH6.8~7.4。
第二方面,本发明实施例提供上述任一实施例所述的冻干保护剂的制备方法,包括:
将灭菌后的各组分溶于溶剂中,混匀。
第三方面,本发明实施例提供上述任一实施例所述的溶瘤病毒冻干粉的制备方法,包括:
将溶瘤病毒与上述任一实施例所述的冻干保护剂混合后再冻干。
影响冻干保护剂保护性能的因素主要来自两方面:一是微生物因素,微生物的种类和结构差异、培养方法的选择以及微生物的种类(如溶瘤病毒,冻干后要求依然保有肿瘤杀伤作用)等;二是保护剂的配方,选用保护剂的原料及其性质、原料配比及与该疫苗适应的冻干曲线等。在选择冻干保护剂的组方时,要考虑上述几种主要因素,选择合适的组方与搭配,并通过反复验证制定冻干曲线。
在一些实施方式中,所述冻干的方法包括:
预冻、一次干燥和二次干燥三个主要步骤。
第四方面,本发明实施例提供上述任一实施例所述的溶瘤病毒冻干粉的制备方法制备得到的溶瘤病毒冻干粉。
在本申请中,所用的溶瘤病毒可以选自ssDNA类病毒、dsDNA类病毒、ssRNA类病毒或dsRNA类病毒;和/或,所用的溶瘤病毒可以选自野生型病毒株或自然减毒株、基因工程选择性减毒株、基因加载型病毒株或基因转录靶向型病毒株。
所述野生型病毒株或自然减毒株可以选自新城疫病毒、呼肠孤病毒、流行性腮腺炎病毒、西尼罗河病毒、腺病毒、牛痘病毒等。
所述基因工程选择性减毒株可以通过人工方式删除关键基因而实现病毒复制的肿瘤选择性,所述基因工程选择性减毒株例如ONYX-015、G207。
所述基因加载型病毒株可以加载外源基因,所述外源基因例如为粒细胞巨噬细胞集落刺激因子(GM-CSF),所述基因加载型病毒株例如为JX-594或T-VEC。
所述基因转录靶向型病毒株,即在病毒必需基因前***组织或肿瘤特异性启动子来控制溶瘤病毒在肿瘤细胞内复制,所述基因转录靶向型病毒株例如为G92A。
所述ssDNA类病毒可以选自细小病毒(parvovirus),所述细小病毒例如为H-1PV病毒。
所述dsDNA类病毒可以选自单纯疱疹病毒(herpes simplex virus)、腺病毒(adeno virus)、poxvirus;优选地,所述腺病毒选自Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin(OBP-301),所述单纯疱疹病毒优选为I型单纯疱疹病毒HSV-1;所述单纯疱疹病毒选自R3616、T-VEC、HF10、G207、NV1020、OrienX010,所述poxvirus选自Pexa-Vec(vaccinia virus)、JX-594(vaccinia virus)、GL-ONC1、Myxoma。
所述ssRNA类病毒可以选自Picornavirus、alphavirus、Retroviruses、Paramyxoviruses、Rhabdoviruses;优选地,所述Picornavirus选自CAVATAK、PVS-RIPO、CVA21(enterovirus)、RIGVIR,所述alphavirus选自M1、Sindbis AR339、Semliki Forestvirus,所述Retroviruses选自Toca511,所述Paramyxoviruses选自MV-NIS、PV701(Newcastle disease virus),所述Rhabdoviruses选自VSV-IFNβ、MG1-MAGEA3、VSV-GP。所述ssRNA类病毒还可选自呼肠孤病毒(reovirus)、柯萨奇病毒(coxsackievirus)、脊髓灰质炎病毒(polio virus)、猪塞内加谷病毒(seneca valley virus)、麻疹病毒(measlesvirus)、新城疫病毒(newcastle disease virus)、水泡性口炎病毒(vesicularstomatitis virus,VSV)、流感病毒。
所述dsRNA类病毒可以选自Reoviruses;优选地,所述Reoviruses选自Pelareorep、呼肠孤病毒(Reolysin)、牛痘病毒(vaccinia virus)、腮腺炎病毒、人类免疫缺陷病毒(human immunodeficiency virus,HIV)。在本申请中,所述溶瘤病毒可以包含编码用于癌症治疗的治疗性物质的内源或外源序列。例如,所述治疗性物质可以是细胞因子和/或免疫检查点抑制剂。
细胞因子可以包括白介素,如IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、IL-16、IL-17、IL-18、IL-20;干扰素(IFN)-α、IFNβ、或IFN-γ-、肿瘤坏死因子α(TNFα)、CD40L、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)、和粒细胞集落刺激因子(G-CSF)、趋化因子(诸如中性粒细胞激活蛋白(NAP)、巨噬细胞化学引诱物和活化因子(MCAF)、RANTES、和巨噬细胞炎性肽(MIP-1a和MIP-1b)、补体组分及其受体、免疫***辅助分子(例如B7.1和B7.2)、黏附分子(例如ICAM-1、2和3)以及黏附受体分子。
免疫检查点是调节某些类型的免疫***细胞(如T细胞(其在细胞介导的免疫中起核心作用))的蛋白质。虽然免疫检查点有助于在检查中控制免疫应答,但它们也可以防止T细胞杀死癌细胞。免疫检查点抑制剂(或简称“检查点抑制剂”)可以阻断免疫检查点蛋白活性,从而释放免疫***上的“制动器”,并允许T细胞更好地杀死癌细胞。
如本文所用,术语“免疫检查点抑制剂”或“检查点抑制剂”是指完全或部分地减少、抑制、干扰或调节一种或多种检查点蛋白的分子。检查点蛋白调节T细胞激活或功能。
检查点抑制剂可以包括小分子抑制剂,或者可以包括结合并阻断或抑制免疫检查点受体的抗体或其抗原结合片段,或结合并阻断或抑制免疫检查点受体配体的抗体。可以被靶向以便阻断或抑制的示例性检查点分子包括但不限于CTLA-4、PD-L1、PD-L2、PD-1、B7-H3、B7-H4、BTLA、HVEM、GAL9、LAG3、TIM3、VISTA、KIR、2B4(属于CD2分子家族并且在所有NK、γδ和记忆CD8+(αβ)T细胞上表达)、CD160(也称为BY55)、CGEN-15049、CHK 1和CHK2激酶、A2aR和各种B-7家族配体。B7家族配体包括但不限于B7-1、B7-2、B7-DC、B7-H1、B7-H2、B7-H3、B7-H4、B7-H5、B7-H6和B7-H7。检查点抑制剂包括抗体或其抗原结合片段、其他结合蛋白、生物治疗剂或小分子,这些检查点抑制剂结合并阻断或抑制CTLA-4、PD-L1、PD-L2、PD-1、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD 160和CGEN-15049中一种或多种的活性。
第五方面,本发明实施例提供上述任一实施例所述的溶瘤病毒冻干粉在制备用于杀伤肿瘤的药物中的应用。
在一些实施方式中,所述溶瘤病毒针对的所述肿瘤选自肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌或白血病中的至少一种。
所述溶瘤病毒对肿瘤细胞执行以下杀伤机制中的至少一种:直接裂解肿瘤细胞、原位疫苗和远端效应、诱导固有免疫、激发适应性免疫应答、破坏肿瘤血管***和/或改善抑制性微环境。
以下为具体实施例。
实施例1
本申请实施例中的溶瘤病毒包含外源IL-12、IL-15和Fc融合蛋白表达盒。按照以下顺序依次调整冻干保护剂的配方,利用药典记载的噬菌斑法测定冻干前后溶菌病毒的滴度变化和稳定性变化,滴度测定的复溶剂为注射用水。各配方中的“%”表示的是该组分占冻干保护剂的质量百分数。
(1)选择的4组冻干保护剂配方为:
6a:3%蔗糖+1%明胶+1.0%谷氨酸钠+1.0%HSA+EBSS(Earle's平衡盐溶液)
6b:8%蔗糖+0.3%明胶+0.1%谷氨酸钠+0.1%HSA+EBSS+Tris-HCl
7a:3%蔗糖+1%明胶+1.0%谷氨酸钠+1.0%HSA+1.0%精氨酸+EBSS
7b:8%蔗糖+0.3%明胶+0.1%谷氨酸钠+0.1%HSA+0.1%精氨酸+EBSS+Tris-HCl
冻干保护剂的配制方法:
先将冻干保护剂按照配方溶解于纯化水中,分别用玻璃棒搅拌各成分,然后分别定容。然后将定容后的各冻干保护剂装到灭菌中进行灭菌。调整pH值为6.8-7.4。
将溶瘤病毒加入配置好的冻干保护剂,混合均匀,转移至冻干管进行冻干。
使用该4组冻干保护剂配方的溶瘤病毒滴度如表1所示。
表1
由表1可知,4组配方在冻干后滴度虽有降低,但下降均在1.0Log范围以内,说明基本上均稳定,达到预期要求。
(2)配方调整:
A1)4%蔗糖+0.8%明胶+0.6%HSA+0.5%谷氨酸钠+0.5%精氨酸+0.1%甘油+EBSS
A2)7%蔗糖+0.3%明胶+0.2%HSA+0.1%谷氨酸钠+0.1%甘油+EBSS
A3)4%蔗糖+0.8%明胶+0.6%HSA+0.5%谷氨酸钠+0.5%精氨酸+1%尿素+EBSS
A4)7%蔗糖+0.3%明胶+0.2%HSA+0.1%谷氨酸钠+0.1%尿素+EBSS
B1)4%蔗糖+0.8%明胶+0.6%HSA+0.5%谷氨酸钠+0.5%精氨酸+0.1%甘油+EBSS+Tris
B2)7%蔗糖+0.3%明胶+0.2%HSA+0.1%谷氨酸钠+0.1%甘油+EBSS+Tris
B3)4%蔗糖+0.8%明胶+0.6%HSA+0.5%谷氨酸钠+0.5%精氨酸+1%尿素+EBSS+Tris
B4)7%蔗糖+0.3%明胶+0.2%HSA+0.1%谷氨酸钠+0.1%尿素+EBSS+Tris。
冻干保护剂的配制方法:
先将冻干保护剂按照配方溶解于纯化水中,分别用玻璃棒搅拌各成分,然后分别定容。然后将定容后的各冻干保护剂装到灭菌中进行灭菌。调整pH值为6.8-7.4。
将溶瘤病毒加入配置好的冻干保护剂,混合均匀,转移至冻干管进行冻干。
该8组冻干保护剂配方的滴度如表2所示。
表2
初步稳定性研究:
表2中的8组配方均表现出较好的冻干保护效果,配方A1、A2、A3和A4进行了初步的稳定性考察,分别测定在冻干后于37℃保存7天和14天后的滴度变化。分别测定两个冻干批次。结果如表3和表4所示。
表3
表4
由表3和表4可知,这8组配方间并无明显差异,冻干后的样品在37℃条件下具有较好的稳定性。因此,通过工艺开发和优化得到的冻干制剂配方达到了预期要求。
(3)在A1、A2、A3、A4基础上进行配方调整:
a1)6%蔗糖+0.6%明胶+0.1%HSA+0.3%谷氨酸钠+0.1%精氨酸+0.05%甘油+EBSS
a2)6%蔗糖+0.6%明胶+0.1%HSA+0.3%谷氨酸钠+0.05%甘油+EBSS
a3)5%蔗糖+0.5%明胶+0.2%HSA+0.1%谷氨酸钠+0.1%精氨酸+0.5%尿素+EBSS
a4)5%蔗糖+0.5%明胶+0.2%HSA+0.1%谷氨酸钠+0.5%尿素+EBSS。
冻干保护剂的配制方法:
先将冻干保护剂按照配方溶解于纯化水中,分别用玻璃棒搅拌各成分,然后分别定容。然后将定容后的各冻干保护剂装到灭菌中进行灭菌。调整pH值为6.8-7.4。
将溶瘤病毒加入配置好的冻干保护剂,混合,转移至冻干管进行冻干。
该4组冻干保护剂配方的滴度如表5所示。
表5
由表5可知,本次配方修改后,冻干前后滴度差为0.5±0.1Log PFU/ml,37℃稳定性实验中,a1、a2两组14d滴度差分别为0.56Log PFU/ml和0.86Log PFU/ml,a3、a4两组14d滴度差分别为0.25Log PFU/ml和0.24Log PFU/ml,虽然14d稳定性实验4个组都满足预期要求,但a3和a4两组更优,这两组的冻干前后的滴度稳定性和冻干后的热稳定性都优于前面尝试的各组冻干保护剂的结果。
按照上述方法,对a3配方的溶瘤病毒冻干制剂进一步检测冻干前后的滴度,并进行加速稳定性检测,分别测定在冻干后于37℃保存7天、14天、21天和28天后的滴度变化,检测了两个批次。结果如表6和图8所示,a3配方的溶瘤病毒冻干制剂冻干前后滴度基本上没有变化,且在37℃下放置28天后滴度变化较小,仍能稳定存在。说明本发明的冻干配方在长期保存后仍具有良好的稳定性。
表6
滴度(lgPFU/ml) | a3批次1 | a3批次2 |
冻干前 | 7.80 | 7.92 |
冻干后 | 7.70 | 7.79 |
37℃-7d | 7.51 | 7.41 |
37℃-14d | 7.31 | 7.33 |
37℃-21d | 7.29 | 7.16 |
37℃-28d | 7.20 | 7.01 |
实施例2样品冻干前后外源蛋白的表达检测
本实施例用以a3配方为例,使用冻干前后的样品感染Vero细胞,用ELISA法检测样品冻干前和冻干3天后细胞因子IL-12和IL15,以及hIgG4的表达。使用的冻干保护剂配方为两个批次的a3配方:5%蔗糖+0.5%明胶+0.2%HSA+0.1%谷氨酸钠+0.1%精氨酸+0.5%尿素。
Vero细胞铺板至6孔板,计数,根据计数结果(约为1x106cell/ml)调整病毒pfu浓度。用Trypsin消化细胞,吸去孔中培养基,用PBS清洗后,加入DMEM培养基稀释后的冻干病毒液,浓度梯度稀释,将孔板放置在摇床上室温孵育2h。孵育完成后,吸去孔中病毒液,每孔补加2ml 1%FBS的MEM培养基,然后于37℃、CO2培养箱培养72h。
用Duoset Ancitilary rengent kit 2(厂家:RD system,货号:DY008)、HumanIL-12p70 elisa SA kit(厂家:RD system,货号:DY1270)和Human IL-15/IL-15Racomplex(厂家:RD system,货号:DY6924)检测感染病毒后的Vero细胞分泌IL-15和IL-12的量,用Thermo的Multiskan Go酶标仪450nm和570nm读数。数据分析采用Prism8和Excel软件,曲线拟合采用四项式拟合。
结果如下表和图1-图3、表7所示,样品冻干前后感染Vero细胞后,分泌IL-12和IL15的量没有显著变化,说明冻干对病毒样品的活性基本上没有影响。仍能正常表达携带的蛋白。
表7
实施例3样品冻干前后对肿瘤细胞的杀伤效果检测
本实施例使用CCR-8显色法检测样品冻干前后对肝癌细胞Hep 3B的杀伤能力。冻干保护剂与实施例2相同。
复苏、传代Hep 3B细胞,消化,计数,铺板,用培养基调整至细胞悬液浓度为4x105cell/ml,然后以100ul/孔加入96孔板中。将用DMEM培养基梯度稀释冻干前后的病毒样品加入铺好细胞的96孔板中,感染细胞,MOI为20、4、0.8、0.16、0.032、0.006、0.0128、0.0003和0.0000512。置于37℃、CO2培养箱培养72h。用CCK8(厂家:YEASEN,货号:40203ES80)检测感染后的Hep 3B细胞的活性,用用Thermo的Multiskan Go酶标仪450nm和650nm读数。数据分析采用Prism8和Excel软件,曲线拟合采用四项式拟合。
结果如下表和图4-图7、表8所示,
表8
此外,对冻干前后的样品进行支原体(EZ-PCR Mycoplasma Detection Kit,厂家:BI,货号:20-700-20)和内毒素(Toxin SensorTM Chromogenic LAL Endotoxin AssayKit,厂家:金斯瑞,货号:L00350C)检测,在最低检测限内均未检测支原体和内毒素,说明本申请样品具有较高的安全性。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,便于具体和详细地理解本发明的技术方案,但并不能因此而理解为对发明专利保护专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准,说明书可以用于解释权利要求的内容。
Claims (12)
1.一种适用于溶瘤病毒的冻干保护剂,其特征在于,其为具有功能成分的溶液,以工作浓度下的质量百分数计,所述功能成分包括:
蔗糖3%-10%、明胶0.3%-1.0%、人血清白蛋白(HSA)0.1%-1.0%和谷氨酸钠0.1%-1.0%。
2.根据权利要求1所述的冻干保护剂,其特征在于,以工作浓度下的质量百分数计,其还包括精氨酸0.1%-1.0%。
3.根据权利要求1所述的冻干保护剂,其特征在于,以工作浓度下的质量百分数计,其还包括尿素0.1%-1.0%。
4.根据权利要求1所述的冻干保护剂,其特征在于,以工作浓度下的质量百分数计,其还包括甘油0.01%-0.15%。
5.根据权利要求1~4任一项所述的冻干保护剂,其特征在于,以工作浓度下的质量百分数计,所述功能成分由以下组分组成:
蔗糖5%-6%、明胶0.5%-0.6%、HSA 0.1%-0.2%、谷氨酸钠0.1%-0.3%、尿素0.5%-0.6%和精氨酸0.1%-0.15%;或者
蔗糖5%-6%、明胶0.5%-0.6%、HSA 0.1%-0.2%、谷氨酸钠0.1%-0.3%和尿素0.5%-0.6%;或者
蔗糖5%-6%、明胶0.5%-0.6%、HSA 0.1%-0.2%、谷氨酸钠0.1%-0.3%和甘油0.05%-0.06%;或者
蔗糖5%-6%、明胶0.5%-0.6%、HSA 0.1%-0.2%、谷氨酸钠0.1%-0.3%、甘油0.05%-0.06%和精氨酸0.1%-0.15%。
6.根据权利要求1~4任一项所述的冻干保护剂,其特征在于,所述溶液的溶剂为水。
7.根据权利要求1~4任一项所述的冻干保护剂,其特征在于,所述冻干保护剂还包括缓冲物质,所述冻干保护剂的工作酸碱度为pH6.8~7.4。
8.权利要求1~7任一项所述的冻干保护剂的制备方法,其特征在于,包括:
将灭菌后的各组分溶于溶剂中,混匀。
9.一种溶瘤病毒冻干粉的制备方法,其特征在于,包括:
将所述溶瘤病毒与权利要求1~7任一项所述的冻干保护剂混合后再冻干。
10.权利要求9所述的方法制备得到的溶瘤病毒冻干粉。
11.根据权利要求10所述的溶瘤病毒冻干粉,其特征在于,所述溶瘤病毒选自DNA病毒或RNA病毒。
12.如权利要求10~11任一项所述的溶瘤病毒冻干粉在制备用于杀伤肿瘤的药物中的应用。
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