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铜绿假单胞菌外毒素 A 在生物制药中应用的研究进展

2020-05-26 14:32 出处:未知 人气: 评论(0
  摘要:铜绿假单胞菌外毒素A(Pseudomonas aeruginosa exotoxin A,PEA)是该菌分泌的外毒素之一,通过催化细胞延伸因子2(elongation factor-2,EF-2)的ADP核糖基化,抑制蛋白合成,从而导致细胞死亡,在铜绿假单胞菌导致的感染性疾病中发挥重要作用。PEA强烈的细胞毒性特点使得该毒素成为免疫毒素的重要成分之一,已广泛应用于肿瘤的靶向治疗中。此外,由于PEA可与抗原提呈细胞表面的α2巨球蛋白受体结合,通过胞饮作用进入胞内,辅助抗原的处理和提呈,可作为分子佐剂应用于各种疫苗的研发。本文对PEA在免疫毒素及疫苗佐剂方面应用的研究进展作一综述。
 
  关键词:铜绿假单胞菌外毒素A;生物制药;应用
 
  铜绿假单胞菌是一种常见的条件致病菌[1-3]。许多免疫力低下患者常并发严重的铜绿假单胞菌感染[3-5],表现为细胞坏死、脂肪性变、肾出血、低血压性休克等,其中以肝细胞坏死最为严重。1972年,LIU在临床分离株的培养液中,首次发现了导致铜绿假单胞菌感染的毒性因子—外毒素A,并指出其可引起皮肤黏膜坏死、诱发败血症等[6-7]。大多数革兰阴性菌的内毒素毒性较强,而铜绿假单胞菌恰恰相反,其外毒素A的毒性最强,能够对机体组织及器官造成极大危害。
 
  铜绿假单胞菌外毒素A(Pseudomonas aeruginosa exotoxin A,PEA)由铜绿假单胞菌的toxA基因编码,翻译成蛋白后,通过细菌的Ⅱ型分泌系统分泌至胞外。PEA为单链蛋白,全长613个氨基酸,相对分子质量约66 000,由信号肽和功能区构成。其功能区由3个结构域组成,从N-末端到C-末端依次为Ⅰ、Ⅱ和Ⅲ区。Ⅰ区分为Ⅰa(Met1-Glu252)和Ⅰb(Gly365-Glu399)两部分,其中Ⅰa区是结合区,主要与靶细胞表面受体相结合;Ⅰb区的部分残基(Ser385-Gly404)与PEA的酶活性相关。Ⅱ区(Gly 253-364 Asn)主要由6个连续的α螺旋构成,含有关于蛋白分泌和转位信息,负责将有活性的毒素片段转移至细胞质,是毒素的转位相关结构域。PEA的ADP核糖基化酶活性区域是其发挥细胞毒性的关键功能区,由Ⅲ区的氨基酸残基(Gly 405-Lys 613)和部分Ⅰb区的残基(Ser 385-Gly 404)组成。结构域中8个半胱氨酸形成4个稳定的二硫键,在细胞质内催化EF-2的ADP核糖基化,阻止EF-2促进肽键延长作用,从而抑制蛋白合成,导致细胞死亡。其中Glu 553位于活性中心,是PEA细胞毒性的关键残基,通过催化EF-2的核糖基转移发挥作用[8-11]。
 
  1外毒素A在免疫毒素(immunotoxins,ITs)中的应用
 
  ITs又称生物导弹,是将具有细胞毒性的分子(毒素)和具有导向能力的分子(载体)偶联而成的具有特异性杀伤能力的生物制剂[12-16]。将PEA与靶向分子融合可以获得相应的免疫毒素,通过靶向分子将PEA结合至肿瘤细胞,在目标细胞内发挥PEA的细胞致死活性,导致肿瘤细胞死亡,从而达到治疗肿瘤的目的[17-20]。重组免疫毒素常用的铜绿假单胞菌外毒素为PE40(缺失Ⅰa区的PEA片段)。将PE40与特异性抗体连接制成重组毒素,对受体细胞具有强烈的靶向杀伤性。目前已经有13个PEA为基础的免疫毒素处于临床试验阶段,应用前景广阔[21]。但此类免疫毒素对实体瘤穿透率低,体内半衰期短,因此,研究人员通过应用基因工程手段改造铜绿假单胞菌外毒素分子(如PE38、PE38KDEL、PE35等突变体)来解决这些问题(见表1)。以下对已进入临床阶段的PEA免疫毒素进行简要介绍。
  1.1 SS1P SS1P即SS1(Fv)-PE38,是一种重组的治疗性蛋白,由高亲和力的二硫键Fv组成,可靶向至负载于PEA的间皮素上[34-37]。目前已完成Ⅰ期临床试验。间皮素是一种细胞表面糖蛋白,可表达于多种实体瘤,如间皮瘤、卵巢癌和胰腺癌[35,38-44]。有研究纳入患有间皮瘤、卵巢瘤、胰腺癌的24名患者,连续10 d每天输注SS1P,间隔4周重复1次。24名患者中18名(75%)检测到免疫原性,其中5名(21%)接受了2轮治疗。在为期10 d的输注中,可检测到稳定的SS1P血药浓度,中位峰值水平达153 ng/mL。试验结果显示了良好的临床活动证据,且这些含有大量单克隆抗体的化学共轭物可延长药物在血浆中的半衰期[45]。
 
  1.2 BL22 BL22又称RFB4(dsFv)-PE38,由抗CD22单克隆抗体的可变区与相对分子质量38 000的PEA片段融合而成[46-47]。在Ⅰ期和Ⅱ期人体临床试验中,BL22可诱导成人毛细胞白血病(对嘌呤类似物耐受)的完全缓解且不影响个体持续发育。在患有CD22+急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)和霍奇金淋巴瘤(non-Hodgkin lymphoma,NHL)儿童中,每次用药量10~40μg/kg,每日3~6次,间隔21~28 d重复1次。结果显示,大部分受试者均产生短暂的临床疗效,抗CD22抗体具有明显的靶向效果[48]。
 
  2外毒素A作为疫苗佐剂的应用
 
  与霍乱毒素、大肠埃希菌不耐热肠毒素类似,PEA的类毒素及无毒突变体也是一个良好的候选佐剂。研究发现,PEA可携带抗原进入局部黏膜、肝、脾和淋巴结等抗原提呈细胞内,直接辅助抗原特别是Ⅰ类主要组织相容性复合体(major histo-compatibility complex-Ⅰ,MHC-Ⅰ)类抗原肽的处理和提成。目前,已经有超过43种抗原均尝试使用化学偶联、基因工程融合等方式,利用PEA作为佐剂以提高免疫效果(表2),但目前尚未见有上市的以PEA作为佐剂的疫苗。下面就以广谱甲型流感病毒疫苗、疟疾疫苗为例,介绍PEA在佐剂研究中的应用。
  2.1广谱甲型流感病毒疫苗甲型流感病毒是一种引起急性呼吸道传染性疾病的单股负链RNA病毒[63]。病毒膜蛋白M2的氨基酸序列保守,且其抗血清具有抑制流感病毒复制的功能。但M2蛋白胞外区(M2 extracellular domain,M2e)仅由24个氨基酸残基组成,免疫原性较弱。PEAⅢ区中的第553位谷氨酸缺失后因失去了ADP核糖基酶活性,变成无毒形式ntPEA,但这种突变体仍能与天然毒素竞争结合靶细胞受体。因此,可用ntPEA为分子佐剂,以增强疫苗的免疫原性。有研究通过构建甲型流感病毒M2蛋白胞外区与PEA融合蛋白的原核表达载体,将其表达产物胶回收后与弗氏不完全佐剂联合皮下免疫BALB/c小鼠,终免2周后,用5个LD50流感病毒A/PR/34/8株进行攻毒。结果表明,免疫组可诱导小鼠产生抗M2e特异性抗体并能够抑制病毒在肺内的复制[51]。
 
  2.2疟疾疫苗为增强疟疾候选疫苗的免疫原性,将红细胞前期的顶端膜抗原1(apical membrane an-tigen 1,AMA1)疫苗和针对蚊体内发育阶段的表面蛋白25(Pfs25)疫苗,均与无毒重组的PEA(recombinant nontoxic PEA,rPEA)化学共轭结合,构建重组工程疫苗[64]。有研究在0及28 d分别免疫小鼠,AMA1-rPEA和Pfs25-rPEA与铝佐剂吸附后可刺激产生明显的抗体反应。对功能活性进行检测时,AMA1-rPEA刺激产生的抗体可抑制血液寄生虫对红细胞的侵袭,Pfs25-rPEA刺激产生的抗体可抑制寄生虫在按蚊肠内的发育,表明这种结合不会破坏引发功能性免疫应答所需的重要表位[65]。实验结果证明,rPEA具有良好的佐剂效应。
 
  3展望
 
  近年来,随着基因工程技术的发展以及对PEA结构和功能的深入研究,利用PEA构建重组毒素提高疫苗免疫原性的研究取得了很大进展。相信在不久的将来,研究人员还会开发出更多潜在的功能,让PEA重组免疫毒素、疫苗佐剂更好地发挥其作用。
 
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