CN110996989A - 降低农杆菌内毒素的毒性 - Google Patents
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- CN110996989A CN110996989A CN201880050287.5A CN201880050287A CN110996989A CN 110996989 A CN110996989 A CN 110996989A CN 201880050287 A CN201880050287 A CN 201880050287A CN 110996989 A CN110996989 A CN 110996989A
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Abstract
本发明涉及经遗传修饰的农杆菌菌株,疫苗佐剂以及一般分子生物学和免疫学领域。本文提供了产生毒性降低的脂多糖(LPS)或脱毒脂多糖的经修饰农杆菌菌株以及获取此类菌株以供植物生产生物制品的方法。本文还提供毒性降低或脱毒LPS作为临床适用佐剂的用途。
Description
相关申请的交叉引用
本申请要求2017年6月5日提交的第62/515,141号美国临时申请的优先权,其通过引用全文纳入本文用于所有目的。
背景
脂多糖(LPS)也称为内毒素,是由革兰氏阴性菌产生的。这是一种毒素和热原,会在宿主中引发强烈的免疫反应,生物制品的LPS污染会引起患者高烧和体内促炎性细胞因子生产失控,常常造成败血性休克。所以,FDA等监管机构对生物制品及其他药品中消除LPS毒性有严格要求。
农杆菌介导的遗传转化是最为优选的植物遗传转化方法,因其方法容易实施且效益高。举例来说,农杆菌菌株被广泛用于将靶基因递送到植物细胞中用于生产生物制品。像在其他革兰氏阴性菌中一样,脂多糖(LPS)位于农杆菌的外膜上。LPS不容易从某些植物生产的生物制品(PMB)中去除。尤其,当前从产自植物的药品生产中消除内毒素的方法涉及昂贵且高劳动强度的纯化过程,而这些过程对于许多目标药物而言仍不充分。因此,迫切需要针对PMB中农杆菌源性LPS污染问题的解决方案。
发明概述
为了提供清楚简洁的概述,以下描述涉及某些示例性内容和实施方式。本领域普通技术人员鉴于本申请的教导容易发现并理解本文所述技术会有其他方面内容、实施方式、构造和改变,并且容易发现并理解在此概述中所述或本申请别处所述示例性内容和实施方式既非限制亦非穷举。
第一个方面,本文提供了一种经遗传修饰的农杆菌属菌,所述农杆菌属菌具有合成极长链脂肪酸(VLCFA)所需一种或多种多肽的功能性缺失,并且所述农杆菌属菌的脂多糖(LPS)毒性低于其亲本株。可以通过AcpXL-依赖性脂质A酰基转移酶编码基因(lpxXL)的至少部分去除来实现所述功能性缺失。可以通过酰基载体蛋白编码基因(acpXL)的至少部分去除来实现所述功能性缺失。可以通过lpxXL的至少部分去除和acpXL的至少部分去除来实现所述功能性缺失。某些情形中,经遗传修饰的农杆菌包含编码脂质A 4'-磷酸酶(LpxF)的外源核酸。编码LpxF的外源核酸可以源自图拉弗朗西斯菌(Francisella tularensis),所述农杆菌能够感染或经构造而感染植物细胞、介导T-DNA转入植物细胞以及介导T-DNA***植物细胞基因组。农杆菌菌株可选自根癌农杆菌(Agrobacterium tumefaciens,亦即A.tumefaciens)和发根农杆菌(Agrobacterium rhizogenes)。亲本株可以是根癌农杆菌GV3101或根癌农杆菌LBA4404,并且所述经遗传修饰的细菌保留了介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。亲本株可以是根癌农杆菌(C58),并且所述经遗传修饰的细菌保留了将肿瘤诱导基因递送到植物细胞中的能力。
另一方面,本文提供一种降低农杆菌属菌中脂多糖(LPS)毒性的方法。所述方法可以包括以下所述或主要由以下所述组成:删除或破坏所述细菌的LpxxL编码基因或AcpXL编码基因的至少一部分,由此获得经遗传修饰的农杆菌,所述经遗传修饰农杆菌的LPS毒性低于包含所述被删除或被破坏基因的农杆菌。某些情形中,所述方法还包括导入编码脂质A4'-磷酸酶(LpxF)的外源核酸。所述外源核酸可源自图拉弗朗西斯菌(Francisellatularensis)。所述农杆菌属菌可以是根癌农杆菌菌株GV3101或根癌农杆菌菌株LBA4404,并且所述经遗传修饰的细菌保留了介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。所述农杆菌属菌可以是根癌农杆菌菌株C58,并且所述经遗传修饰的细菌保留了将肿瘤诱导基因递送到植物细胞中的能力。
另一方面,本文提供一种降低农杆菌属菌中LPS毒性的方法。所述方法可以包括以下所述或主要由以下所述组成:将编码LpxF的外源核酸导入细菌,由此获得经遗传修饰的农杆菌,所述经遗传修饰农杆菌的LPS毒性低于不含所述外源核酸的农杆菌。所述编码LpxF的外源核酸可源自图拉弗朗西斯菌(Francisella tularensis)。所述农杆菌属菌可以是根癌农杆菌菌株GV3101或根癌农杆菌菌株LBA4404,并且所述经遗传修饰的细菌保留了介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。所述农杆菌属菌可以是根癌农杆菌菌株C58,并且所述经遗传修饰的细菌保留了将肿瘤诱导基因递送到植物细胞中的能力。
另一方面,本文提供一种产生内毒素毒性降低且免疫原性改善的LPS变体分子的方法,所述方法包括以下步骤:(a)按照本文所述方法修饰农杆菌菌株;和(b)让经修饰的农杆菌菌株在适宜条件下生长从而产生LPS变体分子,所述LPS变体分子的内毒素毒性低于且免疫原性优于未按步骤(a)修饰的农杆菌菌株所产LPS分子。某些情形中,该方法还包括分离所述LPS变体分子。
另一方面,本文提供一种脂多糖(LPS),其分离自本文所述方法所得经遗传修饰的农杆菌,所述LPS的毒性低于野生型农杆菌或未经所述遗传修饰的农杆菌所产LPS。
本文还提供一种佐剂,包含免疫刺激量的本文提供的毒性降低的LPS和药学上可接受的运载体。
本文还提供一种免疫原性组合物,包含编码能够引发免疫反应的抗原的多核苷酸和佐剂,所述佐剂包含免疫刺激量的毒性降低LPS。
附图说明
图1A展示acpXL突变株释放的脂质A的脂肪酸组成分析。显示的是脂质A的气相色谱/质谱(GCMS)图。注意结果中没有VLCFA。
图1B展示acpXL/lpxF突变株释放的脂质A的脂肪酸组成分析。显示的是脂质A的气相色谱/质谱(GCMS)图。注意结果中没有VLCFA。
图2展示acpXL突变株的MALDI-TOF谱。插图:突变株的主要脂质A物质。R1在四乙酰化簇中可以是H+,或在五乙酰化簇中可以是16:0、18:1。R2在四乙酰化簇中可以是H+,或在五乙酰化簇中可以是b-OHC14:0。
图3显示本氏烟草(Nicotiana benthamiana)植株上C58变体形成的植物冠瘿样(crown gall-like)肿瘤。将亲本C58或acpXL、acpXL/lpxF根癌农杆菌变体注入本氏烟草的枝干,以MES缓冲液作为阴性对照。农杆菌注射后30天监测肿瘤形成并拍照。
图4显示本氏烟草植株叶中绿荧光蛋白(GFP)的表达。将GFP基因转入亲本株GV3101、acpXL和acpXL/lpxF根癌农杆菌菌株。然后将根癌农杆菌或仅MES缓冲液(阴性对照)渗入到本氏烟草叶的不同区域。农杆菌渗入后4天测定GFP表达。
具体说明
本文提供的组合物和方法至少部分基于发明人开发了对脂质A合成所涉及多肽的编码基因的修饰;开发出的修饰改变了脂多糖(LPS)的毒性。LPS是具有独特刺激机制的强免疫反应活化剂。然而,由于炎性免疫反应的过度激活,LPS对人类和动物具有固有毒性。LPS主要有三个结构域:脂质A、核心寡糖和O-抗原。脂质A组分是LPS的有效毒性部分,负责革兰氏阴性菌的LPS免疫刺激活性。不囿于任何特定理论和作用模式,据信,含有四酰化或五酰化或单磷酸化脂质A物质的农杆菌LPS表现出显著降低的毒性以及降低的、相当的或改善的免疫原性。由于农杆菌生产包含四酰基或五酰基或单磷酸化脂质A物质的LPS,且农杆菌是植物致病菌而不是人或动物细胞的病原体,因此毒性降低的LPS适合用作内毒素毒性风险降低或消除且没有(或基本上没有)人类致病菌的细菌组分或副产物污染的佐剂。
本文提供的经遗传修饰的农杆菌具有多重优点。举例来说,LPS毒性降低或表达脱毒LPS的农杆菌变体其优点在于生产更安全的用于人和非人动物的植物性生物制品。通过在LPS毒素于细菌细胞内产出之前降低或消除其毒性,用包含变异农杆菌的细胞产生的生物分子对昂贵且费力的纯化过程的需要减少或消除,所述变体加速了药物获准、提高了药物安全性并且降低了生物药生产的成本。对佐剂生产而言,农杆菌是植物特异性病源,因此用农杆菌生产的佐剂比用人病原性细菌生产的佐剂更安全。因此,文本提供的经遗传修饰的农杆菌适合生产毒性降低或脱毒LPS,毒性降低或脱毒LP是安全有效的佐剂,适合由食品和药物管理局(FDA)批准用于临床。
定义
除非另外定义,本文中的所有技术和科学术语都具有本文所属领域普通技术人员通常所理解的含义。若有冲突,以本说明书(包括定义)为准。除非另作说明,单数术语涵盖复数,复数术语涵盖单数。本文提及的所有出版物、专利和其他参考文献通过援引整体纳入本文用于所有目的,如同单独具体指明每篇出版物或专利申请通过援引纳入,除非指明仅通过援引纳入某专利或专利出版物的特定部分。
为了进一步阐明本文公开的内容,提供以下术语、缩写和定义。
如本文所用,术语“包含”、“包括”、“(具)有”、“含有”以及它们的变换形表示非排他性开放式限定。例如,包含一系列元素的组合物、混合物、过程、方法、物品或设备不一定仅限于那些元素,而可以包括未列明的其他元素或此类组合物、混合物、过程、方法、物品或设备所固有的其他元素。并且,除非另有明确说明,“或(者)”表示兼容性的“或(者)”而非排他性的“或(者)”。例如,以下任何一项满足A或B的情形:A真(或存在)而B伪(或不存在),A伪(或不存在)而B真(或存在),以及A与B均为真(或存在)。
如本文所用,术语“多核苷酸”、“核酸”和“核酸分子”可互换使用,并且涵盖单个核酸;多个和多种核酸;核酸片段、其变体或衍生物;核酸构建体(例如信使RNA(mRNA)和质粒DNA(pDNA))。多核苷酸或核酸可包含全长cDNA序列的核苷酸序列或其片段,包括非翻译5'和/或3'序列以及编码序列。多核苷酸或核酸可包含任意聚核糖核苷酸或聚脱氧核糖核苷酸,可以包括非修饰核糖核苷酸或脱氧核糖核苷酸或经修饰的核糖核苷酸或脱氧核糖核苷酸。例如,多核苷酸或核酸可包含单链和双链DNA;单链区双链区混合DNA;单链和双链RNA;单链区双链区混合RNA。包含DNA和RNA的杂合分子可以是单链、双链或单链区双链区混合。这些术语还包括多核苷酸或核酸的化学修饰、酶修饰以及代谢修饰形式。
某具体DNA也指其互补链,即按照脱氧核糖核苷碱基配对规则确定的序列。
如本文所用,术语“基因”指编码功能性产物(RNA或多肽/蛋白质)的核酸。基因包括功能性产物编码序列之前(5′非翻译序列)和/或之后(3′非编码序列)的调控序列。如本文所用,术语“编码序列”指编码特定氨基酸序列的核酸序列。
如本文所用,术语“多肽”包括单个多肽、多个和多种多肽,以及多肽的片段。该术语指包含通过酰胺键(亦称肽键)线性相连单体(氨基酸)的分子。术语“多肽”指两个或更多氨基酸构成的任意一条或多条链,并不表示特定长度或大小的产物。因此,肽、二肽、三肽、寡肽、蛋白质、氨基酸链以及用于指示两个或更多氨基酸构成的一条或多条链的其他术语都包含在“多肽”的定义内,且这些术语与“多肽”在本文中互换使用。多肽可从天然生物来源中分离或由重组技术产生,但某特定多肽不一定由某特定核酸序列翻译得来。多肽可以任意合适的方式产生,例如包括但不限于化学合成。
如本文所用,术语“修饰”可以指本文所述多核苷酸内造成该多核苷酸所编码多肽的活性降低、基本消除或消除的改变(例如破坏),以及本文所述多肽内造成该多肽活性降低、基本消除或消除的改变。或者,术语“修饰”可以指本文所述多核苷酸内造成该多核苷酸所编码多肽的活性升高或增强的改变,以及本文所述多肽内造成该多肽活性升高或增强的改变。这些改变可以通过本领域所知的方法来制造,包括但不限于删除、突变(例如自发突变、随机突变、由增变基因(mutator gene)引起的诱变或转座子诱变)、取代、***、下调、改变细胞定位、改变多核苷酸或多肽的状态(例如甲基化、磷酸化或泛素化)、去除辅因子、导入反义RNA/DNA、导入干扰RNA/DNA、化学修饰、共价修饰、UV或X射线照射、同源重组、有丝***重组、启动子置换法和/或其组合。
如本文所用,术语“经遗传修饰(的)”和“经遗传工程化(的)”互换使用,指原核或真核细胞经修饰而包含人工创制或修饰过的(例如采用重组DNA技术)非天然核酸分子或由其衍生(例如转录、翻译等)所得的非天然核酸分子。包含外源、重组、合成和/或其他方式修饰的多核苷酸的农杆菌属菌被认为是经遗传修饰的细胞,因此相对于任何天然存在的对应农杆菌属菌而言是非天然存在的。某些情形中,经遗传修饰的细胞含有一种或多种重组核酸。另一些情形中,经遗传修饰的细胞含有一种或多种合成的或经遗传工程化的核酸(例如相对于其天然对应细胞含有至少一处人工***、缺失、倒置或取代的核酸)。产生遗传工程化细胞的方法是本领域所知的,可见Sambrook等人的《分子克隆,实验室手册》(MolecularCloning,A Laboratory Manual),冷泉港出版社,冷泉港,纽约(1989),在此导入作为参考。
术语“表达”在此指基因产物的生物合成。例如,就结构基因而言,表达包括将结构基因转录成mRNA,以及—可选地—随后将mRNA翻译成一种或多种多肽。术语“表达”也指源自DNA的有义(mRNA)或反义RNA的转录和稳定积累。如本文所用,术语“过度表达”指高于相同基因或相关基因内源性表达的表达。因此,如果异源基因的表达高于相应内源基因的表达则该异源基因“过度表达”。
术语“农杆菌(Agrobacterium)”在此指土壤传播的革兰氏阴性杆状植物病原菌。农杆菌与根瘤菌(Rhizobium)、中华根瘤菌(Sinorhizobium)和别生根瘤菌(Allorhizobium)同属于根瘤菌科(Rhizobiaceae)(Kersters和De Ley.1984),已根据核糖体特征将其归入变形菌门(Proteobacteria)的α-2亚类(Willems和Collins.1993)。根据16S rDNA比较分析,农杆菌、根癌农杆菌(Agrobacterium tumefaciens)(同放射形农杆菌(Agrobacterium radiobacter))、发根农杆菌(Agrobacterium rhizogenes)、悬钩子农杆菌(Agrobacterium rubi)和葡萄农杆菌(Allorhizobium undicola)与所有根瘤菌(Rhizobium)共同构成一个单系群(Sawada 1993,Young 2003)。
组合物
首先,本文提供了表现出LPS毒性降低或具有脱毒LPS的经遗传修饰的农杆菌属菌。某些实施方式中,农杆菌属菌经遗传修饰而生产含有四酰化脂质A或五酰化脂质A的LPS,其中酰基链之一短于非修饰农杆菌菌株的酰基链。这种情况下,农杆菌属菌经遗传修饰而功能性缺失了脂质A合成所涉及的多肽。根癌农杆菌菌株C58和其他根瘤菌科菌种中主要脂质A组分的结构是本领域所知的(Silipo等,Glycobiology(2004)14(9):805-815;Castro等,Carbohydrate Res.(2008)343:1924-1933)。脂质A是结构高度异质但相当保守的糖脂,通常具有2-脱氧-2-氨基葡萄糖(葡糖胺,GlcN)二糖主链,1位和4'位磷酸化(Raetz等,Annu.Rev.Biochem.71:635-700(2002);Zahringer等,(1999)D.C.Morrison,H.Brade,S.Opal和S.Vogel(编),内毒素与健康和疾病(Endotoxin in health and disease),Marcel Dekker,纽约,第93-114页)。双磷酸化的葡糖胺二糖主链被5个酰基链(五酰化)修饰:酯中的两个未取代14:0(3-OH)脂肪酸,酰胺键中的两个16:0(3-OH),GlcN II上的一个被长链脂肪酸28:0(27-OH)O-酰化(称为极长链脂肪酸(VLCFA))。VLCFA是碳(C)链长大于20的脂肪酸,其进而被3-羟基-丁酰基残基在其羟基上酯化。
某些情形中,通过删除或破坏(例如编码序列突变)至少部分acpXL基因来实现功能性缺失,经遗传修饰的细菌因此不表达功能性AcpXL多肽。acpXL基因(SEQ ID NO:1)编码C28-酰基载体蛋白AcpXL。该多肽是农杆菌中VLCFA合成所必需的。包含acpXL缺失的经修饰农杆菌可生产没有VLCFA的四酰化脂质A。某些情形中,包含acpXL缺失的经修饰农杆菌可产生五酰化脂质A新变体,其中的VLCFA被较短的酰基链(16:0或18:1)取代,或是四酰化和五酰化脂质A的混合物(图1和图2)。
某些情形中,通过删除或破坏(例如编码序列突变)至少部分lpxXL基因、或至少部分lpxXL基因和至少部分acpXL基因(即获得双突变体)来实现功能性缺失,经遗传修饰的细菌因此不表达功能性LpxXL和/或AcpXL多肽。Lpxxl基因编码C28-酰基转移酶(SEQ ID NO:2)。这些多肽是农杆菌中VLCFA合成和转移所必需的。
某些情形中,在野生型(WT)根癌农杆菌C58菌株中制造遗传修饰。另一些情形中,在根癌农杆菌菌株GV3101或LBA4404中制造遗传修饰,此两者是用于将转基因递送到植物细胞中以供生物技术之用的工程化菌株。据细胞检测测定,lpxXL和acpXL基因之一或两者缺失或被破坏的WT菌株和工程化菌株中LPS毒性均显著降低。此外,根癌农杆菌C58菌株中的acpXL变体保留了将肿瘤诱导基因递送到植物中的能力,在烟草叶上产生植物冠瘿样结构的能力被保留证明了这一点(图3)。根癌农杆菌GV3101菌株中的AcpXL变体保留了将转基因递送到植物中的能力(例如介导T-DNA转入植物细胞和T-DNA***植物细胞基因组的能力)。例如,当编码生物分子的转基因用所述经遗传修饰的农杆菌来递送时,能够强劲表达多肽及其他生物分子,如单克隆抗体、病毒样颗粒疫苗和可检测报告蛋白(例如绿荧光蛋白及其变体)(图4)。
某些情形中,农杆菌属菌经遗传修饰而生产含有单磷酸化脂质A的LPS。这种情况下,农杆菌属菌经遗传修饰导入编码脂质A 4'-磷酸酶(LpxF)的外源性lpxF基因,这种4'-磷酸酶使脂质IV(A)(脂质A的四酰化前体)和五酰化脂质A的4'碳(即4'-磷酸部分)上的磷酸基团去磷酸化,但不从六酰化脂质A(例如大肠杆菌中所见)上去磷酸化。LpxF不使脂质A和脂质A前体的1'-磷酸基团、磷脂酸或磷脂酰甘油磷酸去磷酸化。某些情形中,所述外源lpxF基因源自图拉弗朗西斯菌(Francisella tularensis)中的LpxF编码基因(SEQ ID NO:3;另见SEQ ID NO:4(包含用于农杆菌中表达的ATG))。经遗传修饰表达LpxF的野生型和工程化菌株中LPS的毒性都显著降低。
某些情形中,在野生型(WT)根癌农杆菌C58菌株中导入LpxF表达。另一些情形中,在根癌农杆菌GV3101或根LBA4404中制造这样的遗传修饰。此外,农杆菌C58菌株中的lpxF变体保留了将肿瘤诱导基因递送到植物中的能力,在烟草叶上产生植物冠瘿样结构的能力被保留证明了这一点,而农杆菌GV3101菌株中的lpxF变体保留了将转基因递送到植物中(例如用于生物技术目的)的能力。
一些情形中,将编码LpxF的外源核酸(例如源自图拉弗朗西斯菌(Francisellatularensis)的lpxF基因)***AcpXL编码序列所在的基因组区域,由此产生包含至少部分acpXL基因和至少部分lpxF基因缺失或破坏的农杆菌双突变体。由此方式,编码acpXL的基因被破坏,经遗传修饰的变体不表达VLCVA合成所需的功能性AcpXL多肽而表达LpxF。
某些情形中,在野生型(WT)根癌农杆菌C58菌株中制造产生acpXL/lpxF双突变体的遗传修饰。其它情形中,在工程化根癌农杆菌菌株GV3101或LBA4404中制造产生acpXL/lpxF双突变体的遗传修饰。据细胞检测显示,包含acpXL/lpxF双突变的WT菌株和工程化菌株中LPS的毒性显著降低。此外,根癌农杆菌C58菌株中的acpXL/lpxF双突变体保留了将肿瘤诱导基因递送到植物中的能力,在烟草叶上产生植物冠瘿样结构的能力被保留证明了这一点,而根癌农杆菌GV3101菌株中的acpXL/lpxF双突变体保留了将转基因递送到植物中的能力(图4)(例如介导T-DNA转入植物细胞以及T-DNA***植物细胞基因组的能力)。
虽然本文描述了一些示例性农杆菌菌株,但在此讨论的功能可以相同标准移用于其他农杆菌菌株,例如LPS毒性低下、表达脱毒LPS的菌株或者能够被变成LPS毒性低下的其他农杆菌菌株。例如,可用根癌农杆菌(同放射形农杆菌)、发根农杆菌、悬钩子农杆菌和葡萄农杆菌来生产经修饰的农杆菌。例如,可用于本文所述菌株和方法的其他菌株包括但不限于:根癌农根菌(Agrobacterium tumefaciens)菌株C58、根癌农根菌菌株GV3101、根癌农根菌菌株GV3100、根癌农根菌菌株GV3850、根癌农根菌菌株GV2260、根癌农根菌菌株A136、根癌农根菌菌株A136、根癌农根菌菌株Chry5、发根农杆菌(Agrobacterium rhizogenes)菌株、根癌农根菌菌株EHA101、根癌农根菌菌株EHA105、根癌农根菌菌株MOG101、根癌农根菌菌株649、和、根癌农根菌菌株T37。
所有适用于对农杆菌基因进行遗传修饰的方法都可用来产生本文所述的变体。例如,可通过同源重组来破坏靶基因。如本文所用,术语“重组”指两个DNA分子或RNA分子之间的DNA或RNA节段重排。“同源重组”发生在两个杂交DNA分子之间,这两个DNA分子因各自所含的同源或互补核苷酸序列而杂交。如本文所用,术语“同源重组”指两个DNA碱基序列之间在它们具有相似序列或同源序列的区域中发生的重组。如分子生物学领域的技术人员所知,诸如此类的大重复序列是分子内重组的优选靶标,最终导致DNA缺失及其他重排。
另一方面,本文提供一种脂多糖(LPS),其分离自本文所述方法所得经遗传修饰的农杆菌菌株,所述分离的LPS其毒性低于野生型农杆菌菌株或未经所述遗传修饰的农杆菌所产LPS。此类毒性降低的LPS非常适合佐剂之用。例如,经修饰农杆菌菌株产生的毒性降低的LPS可以像药学上可接受的运载体一样以药物组合物的形式来提供。
某些情形中,所述佐剂包含免疫刺激量的毒性降低的LPS和药学上可接受的运载体。如本文所用,术语“免疫刺激”指某种物质(例如本文所述经修饰农杆菌菌株产生的毒性降低的LPS)在动物中激发或诱导免疫原性反应的能力。本文可用的药物组合物还含有药学上可接受的运载体,包括所有合适的稀释剂或赋形剂,这包括本身不诱导对该组合物接受者有害的抗体产生并且其给药没有过度毒性的所有药剂。药学上可接受的运载体包括但不限于诸如水、盐水、甘油和乙醇之类的液体,包括可用于形成喷雾剂用于鼻腔和其他呼吸道递送或眼科***递送的运载体。关于药学上可接受的运载体、稀释剂和其他赋形剂的全面论述可见《雷明顿药物科学》(REMINGTON'S PHARMACEUTICAL SCIENCES)(马克出版公司,新泽西州,新版)。
某些情形中,毒性降低的含LPS佐剂与能够引发免疫反应的抗原或编码抗原的多核苷酸一起提供。可用于本文所述组合物的抗原可源自细胞、细菌或病毒颗粒、或它们的部分。如本文所用,“抗原”指在动物(例如哺乳动物、鸟类或鱼类)中引起免疫原性反应的蛋白质、肽、多糖、糖蛋白、糖脂、核酸或其组合。如本文所述,免疫原性反应可以是体液介导或是细胞介导的。如果免疫原性反应所针对的材料其抗原性较弱,则可以用标准的共价结合技术例如采用多种市售试剂盒将其与载体(例如白蛋白)或半抗原偶联。抗原的例子包括病毒蛋白,例如流感蛋白和乙型肝炎蛋白;细菌蛋白和脂多糖,例如革兰氏阴性细菌细胞壁和表面蛋白。
佐剂还可根据本领域技术人员所知的方法与抗原共价偶联,通常通过抗原上的氨基或羧基与佐剂上一个或多个侧基之间的共价键偶联。尽管在优选实施方案中疫苗组合物的佐剂和抗原是同时给药的,但在另外的实施方案中,佐剂和抗原分开给药至同一部位或临近部位。佐剂的作用是将免疫***的细胞吸引至某部位然后在此作用于抗原。
免疫原性组合物可以作为疫苗通过本领域技术人员已知的引起免疫反应的任何方法施用,包括肠胃外,口服或通过跨膜或透粘膜施用。
方法
另一方面中,本文提供一种降低农杆菌属菌中脂多糖(LPS)毒性的方法。总体说来,这些方法包括删除或破坏编码在LPS合成途径中(包括LPS全部三个结构域:脂质A、核心寡糖和O-抗原的合成)发挥一定功能的蛋白质的基因,或导入在LPS合成途径中有功能的其他细菌的基因,或者既删除LPS合成途径的基因又导入新的LPS合成途径基因。
某些实施方式中,所述方法包括删除或破坏所述细菌的LpxxL编码基因或AcpXL编码基因的至少一部分,由此获得LPS毒性低于包含所述被删除或被破坏基因的农杆菌的经遗传修饰的农杆菌。某些情形中,所述方法包括在农杆菌中导入编码脂质A 4'-磷酸酶(LpxF)的外源核酸。优选的,编码LpxF的外源核酸源自图拉弗朗西斯菌(Francisellatularensis)。可以在根癌农杆菌菌株GV3101或根癌农杆菌菌株LBA4404中制造所述遗传修饰,经遗传修饰的细菌保留介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。另一些情形中,可在根癌农杆菌菌株C58中制造所述遗传修饰,经遗传修饰的细菌保留将肿瘤诱导基因送入植物细胞的能力。
另一方面,本文提供一种降低农杆菌属菌中LPS毒性的方法。某些实施方式中,所述方法包括将编码LpxF的外源核酸导入细菌,由此获得经遗传修饰的农杆菌,所述经遗传修饰的农杆菌的LPS毒性低于不含所述外源核酸的农杆菌。所述外源核酸可源自图拉弗朗西斯菌(Francisella tularensis)。可在根癌农杆菌菌株GV3101或根癌农杆菌菌株LBA4404中制造所述遗传修饰,经遗传修饰的细菌保留介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。另一些情形中,可在根癌农杆菌菌株C58中制造所述遗传修饰,经遗传修饰的细菌保留将肿瘤诱导基因送入植物细胞的能力。
另一方面,本文提供一种产生内毒素毒性降低且免疫原性改善的LPS变体分子的方法,所述方法包括以下步骤:(a)按照本文所述方法修饰农杆菌菌株;和(b)让经修饰的农杆菌菌株在适宜条件下生长从而产生LPS变体分子,所述LPS变体分子的内毒素毒性低于且免疫原性优于未按步骤(a)修饰的农杆菌菌株所产LPS分子。优选的,所述方法还包括将变体LPS与经遗传修饰的农杆菌菌株分离。LPS分子可用常规手段来分离。某些情形中,LPS是从经遗传修饰农杆菌的指数期培养物中提取的。提取的LPS可按照本领域熟知的程序来纯化和定量。如本文所述,从经遗传修饰农杆菌分离的LPS表现出的毒性低于而免疫原性优于野生型农杆菌或未经遗传修饰的农杆菌所产的LPS。
某些情形中,佐剂包含免疫刺激量的按照本文所述方法所得毒性降低的LPS和药学上可接受的运载体、赋形剂或载剂来提供液体制剂。另外,赋形剂或载剂中可含有辅料,例如润湿剂或乳化剂、pH缓冲物质等。这些运载体、赋形剂、载剂和辅料通常是不会在该组合物接受者中诱导免疫反应且其给药无过度毒性的药剂。药学上可接受的运载体和赋形剂包括但不限于诸如水、盐水、聚乙二醇、透明质酸、甘油和乙醇等液体。
某些情形中,佐剂用来制备免疫原性组合物用于疫苗的递送。免疫原性组合物可以包含能够引起免疫反应的抗原(例如多肽、编码抗原的多核苷酸或两者的组合)以及作为佐剂的免疫刺激量的毒性降低的LPS。
另一方面,本文提供用本文所述内毒素毒性降低的农杆菌生产植物性生物制品的方法。某些情形中,所述方法采用“农杆菌渗入法”将携带外源DNA的农杆菌递送到植物组织(例如植物叶)的细胞内空间,从而让外源核酸送入植物细胞基因组中。
产生基于病毒样颗粒的疫苗:VLP与病毒相似但因为它们不含病毒遗传物质而不具有感染性。诸如被膜或衣壳之类病毒结构蛋白的表达可导致病毒样颗粒(VLP)的自我组装。VLP可用作疫苗,因为它们重复、高密度地展示病毒表面蛋白,所述病毒表面蛋白呈现出构象性病毒表位,由此引起强烈的T细胞和B细胞免疫反应。由于VLP不能复制,它们提供了比减毒病毒更安全的替代方案。已在植物中成功产生了不同病毒来源的VLP。然而,由于VLP的动态结构(可以用其小孔呼吸)LPS会陷在VLP内难以去除。这种情况下,可以将可形成VLP的衣壳或包膜蛋白基因(例如乙型肝炎核心抗原HBcAg或诺如病毒衣壳蛋白NVCP)克隆到植物表达载体中,然后此前所述(Santi等,Vaccine,26(15),1846-1854(2008))通过电穿孔法转入根癌农杆菌GV3101或LBA4404菌株。然后培养成本氏烟草植株,用含有HBcAg 3'模块(pICH11599-HBcAg)及其5'TMV模块(用于ER靶向的pICH20999)的低内毒素毒性GV3101或LBA4404菌株和先前所述的整合酶构建体(Chen,2013,用于诺如病毒胃肠炎的病毒样颗粒疫苗(Virus-like Particle Vaccines for Norovirus Gastroenteritis),,刊于M.Giese(编),Molecular Vaccines(第1卷,第153-181页).越南:施普林格公司(Springer);Chen等,Advanced Technolgy in Biology and Medicine,1(1),103-112(2013);Lai和Chen,2012;Lai等,Plant Cell Reports,31(3),573-584(2010);Leuzinger等,Journal ofVisualized Experiments(77)(2013))对本氏烟草植株进行农杆菌共渗透。然后可以在农杆菌渗透后7-10天收获植物的叶,用先前所述的方法纯化VLP(参见Lai和Chen,Immunotherapeutics,9(1),26-49(2013))。
产生抗传染病和癌症的基于抗体的治疗剂:单克隆抗体(mAb)作为重磅抗癌药已经非常成功,并且正在开发用于多种传染性疾病。已在植物中成功生产出抗西尼罗河病毒、登革热病毒、埃博拉病毒和各种癌症的单克隆抗体及其衍生物(例如scFv-Fc、双官能单抗和免疫复合物)(Dent等,Journal of General Virology,97(12),3280-3290(2016);He等,PLoS ONE,9(3),e93541(2014);Lai等,Proceedings of the National Academy ofSciences of the United States of America,107(6),2419-2424(2010);Phoolcharoen,Bhoo,等,Plant Biotechnology Journal,9(7),807-816(2011);Phoolcharoen,Dye,等,Phoolcharoen,Dye,等,Proceedings of the National Academy of Sciences of theUnited States of America,108(51),20695-20700(2011))。然而,必须通过昂贵的下游纯化方法来消除农杆菌带入的LPS。这种情况下,可将抗体轻链(LC)和重链(HC)的基因克隆到MagnICON***植物表达载体pICH21595和pICH11599的5'模块中,如先前所述(Dent等,Journal of General Virology,97(12),3280-3290(2016);He等,PLoS ONE,9(3),e93541(2014);Lai等,Proceedings of the National Academy of Sciences of the UnitedStates of America,107(6),2419-2424(2010);Phoolcharoen,Bhoo,等,PlantBiotechnology Journal,9(7),807-816(2011);Phoolcharoen,Dye,等,Phoolcharoen,Dye,等,Proceedings of the National Academy of Sciences of the United Statesof America,108(51),20695-20700(2011))。然后通过电穿孔将植物表达载体分别转化到低内毒素毒性根癌农杆菌GV3101或LBA4404菌株中,如先前所述(Dent等,Journal ofGeneral Virology,97(12),3280-3290(2016);He等,PLoS ONE,9(3),e93541(2014);Lai等,Proceedings of the National Academy of Sciences of the United States ofAmerica,107(6),2419-2424(2010);Phoolcharoen,Bhoo,等,Plant BiotechnologyJournal,9(7),807-816(2011);Phoolcharoen,Dye,等,Phoolcharoen,Dye,等,Proceedings of the National Academy of Sciences of the United States ofAmerica,108(51),20695-20700(2011))。本氏烟草植株在温室中用16/8小时明/暗周期于25℃生长5周。用含有LC和HC 5'模块及各自对应3'模块的低内毒素毒性GV3101或LBA4404菌株和整合酶构建体对植物叶进行农杆菌共渗透,如先前所述(Giritch等,Proceedingsof the National Academy of Sciences of the United States of America,103(40),14701-14706(2006))。然后在渗透后的第4-10天(dpi)收获经农杆菌渗透的本氏烟草叶,并用提取缓冲液(PBS,1mM EDTA,10mg/ml抗坏血酸钠,10μg/ml亮蛋白肽,0.3mg/ml的苯基甲基磺酰氟)用FastPrep机(Bio101)按照制造商的说明匀浆提取总叶蛋白。通过在4℃、14,000xg离心10分钟来澄清粗制植物提取物。可通过三步纯化方案进一步从澄清提取物中纯化单克隆抗体,该方案包括硫酸铵沉淀、蛋白A亲和层析和DEAE-阴离子交换层析,如先前所述(Lai等,Proceedings of the National Academy of Sciences of the United Statesof America,107(6),2419-2424(2010))。
在可食用植物中生产用于口服递送的疫苗:VLP和其他亚单位疫苗也可以在可食用植物例如莴苣中生产以用于口服免疫,这更易于给药并且对于感染粘膜表面的病原体更有效(Chen和Davis,The potential of plants as a system for the development andproduction of human biologics(第5卷)(2016);Kwon和Daniell,Plant BiotechnologyJournal,13(8),1017-1022(2015);Lai,He,Engle,Diamond和Chen,Plant BiotechnologyJournal,10(1),95-104(2012))。农杆菌LPS的存在阻挠了这一策略的应用。这种情况下,可用低内毒素毒性菌株GV3101或LBA4404如先前所述地将亚单位疫苗或其他生物制品的基因递送到可食植物的叶中(Lai等,Plant Cell Reports,31(3),573-584(2012);Liu Clarke等,Plant Biotechnology Journal,doi:10.1111/pbi.12743)。LPS含量低或无LPS的可食植物材料经最少的加工或纯化即可给目标对象喂食(Liu Clarke等,Plant BiotechnologyJournal,doi:10.1111/pbi.12743)。
在植物细胞培养物中生产疫苗、基于抗体的治疗剂和治疗用酶:整株植株之外,农杆菌还可用于将转基因递送到植物培养细胞中,例如烟草BY-2细胞或胡萝卜细胞(Ramloch-Lorenz,Knudsen和Sturm,Plant J,4(3),545-554(1993))用于生产疫苗、单克隆抗体和治疗用酶。例如,胡萝卜细胞产生的人葡糖脑苷脂酶(商品名ELELYSO)已被FDA批准用于治疗I型Gaucher病(Aviezer等,PLoS ONE,4(3),e4792(2009))。正在用含有葡萄糖脑苷脂酶的胡萝卜细胞开发该药的“可饮用”版本(Shaaltiel等,Plant BiotechnologyJournal,13(8),1033-1040(2015))。这种情况下,可用内毒素毒性降低的农杆菌变体来降低药物的总LPS含量。
已通过一个或多个优选的实施方式描述了本发明的前文所述优点以及其他优点,但应理解,除了明确描述的那些方案之外,会有许多等同方案、替代方案、衍变方案和改换方案,这些都术语本发明范围之内。
序列表
<110> 陈强(Chen, Qiang)
杨明(Yang, Ming)
赖华芳(Lai, Huafang)
<120> 降低农杆菌内毒素的毒性
<130> 112624.00984.M17-223L-WO1
<150> 62/515,141
<151> 2017-06-05
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 285
<212> DNA
<213> 根癌农杆菌(Agrobacterium tumefaciens)
<400> 1
atgggcgtga cagctacatt cgacaaggtt gccgacatta tcgccgaaac cagcgaaatc 60
gaccgcgaaa ccattacgcc ggagagccac acgatcgacg atctgggcat cgacagcctc 120
gactttctcg atatcgtttt tgccatcgac aaggaattcg gcatcaagat tccgctcgag 180
cagtggacgc aggaagtcaa cgaaggcaag gtttccaccg aagaatactt cgtgctgaag 240
aacctctgcg ccaagatcga cgaattgcgc gccgccaagg gctga 285
<210> 2
<211> 939
<212> DNA
<213> 根癌农杆菌(Agrobacterium tumefaciens)
<400> 2
ttgaccacct tgaaattatt tctgacgcgg atcgtcctga aactggacca cttccggcag 60
tggctgatcg cgacattcgc ctttggcctt ctcaacctgc tgaagctttt tccggccgat 120
gcgggcattc gcgccacgga ccggctggcg cgcttgatag ggccgaaaac cggccgccac 180
aagctgatgc tctacaatct ggcgcgcgcc tttccggaaa aaaccgaaga ggagcggctg 240
gcgatcgcca tggacagctg ggccaatatg ggccggcttg cggcggaata tgtgtttctc 300
gaccggctgt tcgatttcga cccggaaaag aacgaacccg gccgcatcga agtcgagggt 360
acctcgacct ttctcgaatt gcgcgacaat ccgcggccct tcatcgtttt taccgcccat 420
agcggcaatt tcgaactgct gccggtagca ggctcggcct ttggtcttga tgtgacggtg 480
ctgttccggc cgccgaacaa tccctatgtg gccgacaagg tgttcaattt ccgcaaggaa 540
cgcatgggca atctcgtgcc gtcgcatgcc ggctcctcct tcgcgctggc gcggcaactg 600
gaaaagggcg gcggtgtggg tgtgcttgtc gaccagaagt tcggcaaggg gctgacgacg 660
aagttcttcg ggcttgaggt tcgcaccaac ccgctgcttg ccaagctggt gcggcagttc 720
aattgcgatg tctatcccgc ccgctgcata cgccttccgg acaatcgcta caggctggaa 780
atagagccga aggttgaaat tccccgggat cagaagggca atgtcgatat tcaggcgacg 840
gcgcagcttc tgaacgacaa ggtggaaagc tgggtgcgag aatatcccgg ccaatggctt 900
tggtatcatg atcgctggga cgtgaagcac cagatttga 939
<210> 3
<211> 666
<212> DNA
<213> 图拉弗朗西斯菌(Francisella tularensis)
<400> 3
gcaagatttc atatcatatt aggtttagtt gtttgttttt ttgcatggat attctttctt 60
atattcccga atttggatat acaattcgcg ggacattttt ataattcatc ggcacatcaa 120
tttattggtg ggtatgatgg ctttttagga tttttgcatt ggtttgctag attttttcca 180
atattttttt caataatagt gattttattt ctattaggat cgttatttat cgataagttt 240
aagattaagt atagaaaagc tatattcttt attgcggtat gcttatggat aggtccaggt 300
ttggttgtta actatgtgtt taaagatcat tgggggcgtc caagaccagt gatggtagag 360
caatttaatg gcgacaaaat ttttcaacca ccattcgtta tatcttcaca atgtgataaa 420
aactgctcct ttgtatgtgg tgatgcctca atgggatttt ggctttttgc atttatgcca 480
ttactagcta caagaaaaaa gaagcttgtt gcgtttatcg cagcagtagt tgctggtgga 540
ggtttgggat tgatgagaat gtcgcaagga gggcattttt ttagtgatgt tgttttctgt 600
ggcatatttg tgtatatctc aacctgggtg gtttatgcac taatgtatcg taaaaaagaa 660
tattga 666
<210> 4
<211> 669
<212> DNA
<213> 图拉弗朗西斯菌(Francisella tularensis)
<400> 4
atggcaagat ttcatatcat attaggttta gttgtttgtt tttttgcatg gatattcttt 60
cttatattcc cgaatttgga tatacaattc gcgggacatt tttataattc atcggcacat 120
caatttattg gtgggtatga tggcttttta ggatttttgc attggtttgc tagatttttt 180
ccaatatttt tttcaataat agtgatttta tttctattag gatcgttatt tatcgataag 240
tttaagatta agtatagaaa agctatattc tttattgcgg tatgcttatg gataggtcca 300
ggtttggttg ttaactatgt gtttaaagat cattgggggc gtccaagacc agtgatggta 360
gagcaattta atggcgacaa aatttttcaa ccaccattcg ttatatcttc acaatgtgat 420
aaaaactgct cctttgtatg tggtgatgcc tcaatgggat tttggctttt tgcatttatg 480
ccattactag ctacaagaaa aaagaagctt gttgcgttta tcgcagcagt agttgctggt 540
ggaggtttgg gattgatgag aatgtcgcaa ggagggcatt tttttagtga tgttgttttc 600
tgtggcatat ttgtgtatat ctcaacctgg gtggtttatg cactaatgta tcgtaaaaaa 660
gaatattga 669
Claims (28)
1.一种经遗传修饰的农杆菌属菌株,所述经遗传修饰的菌株具有极长链脂肪酸(VLCFA)合成所需一种或多种多肽的功能性缺失,并且相对于其亲本株脂多糖(LPS)毒性低下。
2.如权利要求1所述的经修饰菌株,所述功能性缺失是通过AcpXL依赖性脂质A酰基转移酶编码基因(lpxXL)的至少部分去除实现的。
3.如权利要求1所述的经修饰菌株,所述功能性缺失是通过酰基载体蛋白编码基因(acpXL)的至少部分去除实现的。
4.如权利要求1所述的经修饰菌株,所述功能性缺失是通过lpxXL的至少部分去除和acpXL的至少部分去除实现的。
5.如权利要求3和4中任一项所述的经修饰菌株,所述经修饰菌株产生缺乏VLCFA的四酰化脂质A。
6.如权利要求3和4中任一项所述的经修饰菌株,所述经修饰菌株产生VLCFA被16:0酰基链或18:1酰基链置换的五酰化脂质A。
7.如权利要求3和4中任一项所述的经修饰菌株,所述经修饰菌株产生四酰化脂质A与五酰化脂质A的混合物。
8.如权利要求1所述的经修饰菌株,还包含编码脂质A 4'-磷酸酶(LpxF)的外源核酸。
9.如权利要求8所述的经修饰菌株,所述编码LpxF的外源核酸源自图拉弗朗西斯菌(Francisella tularensis)。
10.如权利要求1所述的经修饰菌株,所述农杆菌属菌能够感染植物细胞,能够介导T-DNA转入植物细胞,并且能够介导T-DNA***植物细胞基因组。
11.如权利要求1所述的经修饰菌株,所述亲本株选自根癌农杆菌(A.tumefaciens)和发根农杆菌(Agrobacterium rhizogenes)。
12.如权利要求1所述的经修饰菌株,所述亲本株是根癌农杆菌GV3101或根癌农杆菌LBA4404,并且所述经遗传修饰的细菌保留了介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。
13.如权利要求1所述的经修饰菌株,所述亲本株是根癌农杆菌(C58),并且所述经遗传修饰的细菌保留了将肿瘤诱导基因递送到植物细胞中的能力。
14.一种降低农杆菌属菌中脂多糖(LPS)毒性的方法,所述方法包括删除或破坏所述细菌的LpxxL编码基因或AcpXL编码基因的至少一部分,由此获得经遗传修饰的农杆菌,所述经遗传修饰的农杆菌的LPS毒性低于包含所述被删除或被破坏基因的农杆菌。
15.如权利要求14所述的方法,还包括导入编码脂质A 4'-磷酸酶(LpxF)的外源核酸。
16.如权利要求15所述的方法,所述外源核酸源自图拉弗朗西斯菌(Francisellatularensis)。
17.如权利要求14所述的方法,所述农杆菌属菌是根癌农杆菌菌株GV3101或根癌农杆菌菌株LBA4404,并且所述经遗传修饰的细菌保留了介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。
18.如权利要求14所述的方法,所述农杆菌属菌是根癌农杆菌菌株C58,并且所述经遗传修饰的细菌保留了将肿瘤诱导基因递送到植物细胞中的能力。
19.一种降低农杆菌属菌中LPS毒性的方法,所述方法包括将编码LpxF的外源核酸导入细菌,由此获得经遗传修饰的农杆菌,所述经遗传修饰的农杆菌的LPS毒性低于不含所述外源核酸的农杆菌。
20.如权利要求19所述的方法,所述编码LpxF的外源核酸源自图拉弗朗西斯菌(Francisella tularensis)。
21.如权利要求19所述的方法,所述农杆菌属菌是根癌农杆菌菌株GV3101或根癌农杆菌菌株LBA4404,并且所述经遗传修饰的细菌保留了介导T-DNA转入植物细胞和介导T-DNA***植物细胞基因组的能力。
22.如权利要求19所述的方法,所述农杆菌属菌是根癌农杆菌菌株C58,并且所述经遗传修饰的细菌保留了将肿瘤诱导基因递送到植物细胞中的能力。
23.一种产生内毒素毒性降低且免疫原性改善的LPS变体分子的方法,所述方法包括以下步骤:
(a)按照权利要求14-22中任一项所述的方法修饰农杆菌菌株;
(b)让经修饰的农杆菌菌株在适宜条件下生长从而产生LPS变体分子,所述LPS变体分子的内毒素毒性低于且免疫原性优于未按步骤(a)修饰的农杆菌菌株所产LPS分子。
24.如权利要求23所述的方法,还包括分离所述LPS变体分子。
25.一种脂多糖(LPS),其分离自按照权利要求14-22中任一项所述方法所得经遗传修饰的农杆菌,所述LPS的毒性低于野生型农杆菌或未经所述遗传修饰的农杆菌所产LPS。
26.一种佐剂,包含免疫刺激量的权利要求25所述毒性降低的LPS和药学上可接受的运载体。
27.一种免疫原性组合物,包含编码能够引发免疫反应的抗原的多核苷酸和佐剂,所述佐剂包含免疫刺激量的权利要求25所述毒性降低的LPS。
28.用于在动物中诱导免疫原性反应的组合物,包含抗原和免疫刺激量的由权利要求1-13中任一项所述经遗传修饰的农杆菌菌株产生的毒性降低的LPS。
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