新型抗金黄色葡萄球菌肠毒素B双特异性抗体的中和保护作用及其机理

Staphylococcal enterotoxins are produced by staphylococcal and streptococcal exotoxins. Staphylococcal enterotoxin (Staphyloccucal enterotoxins, SEs) is a family of bacterial exotoxins, including SEA, SEB, SEC1-3, SED SEE, the SEF, the SEG et al about 20 serotypes, secreted by Staphylococcus aureus. These toxins are responsible for a number of severe human illnesses, including food poisoning, purulent infections and nosocomial infections. .As a superantigen, SEs can bridge MHC class II molecules on antigen presenting cells and TCR β chain V region on T cells leading to the formation of MHC class II molecules: SEs: TCR ternary complex, which can directly activate T cells and lead to the release of a series of inflammatory cytokines causing high fever, nausea, diarrhea, and toxic shock syndrome (TSS). Due to up to 50% case fatality rate of TSS caused by Staphylococcus aureus enterotoxin B (SEB) and as high as 90% of mortality by SEB infection concurrent with the influenza, SEB was considered to be one of standard (“classic”) biological warfare agents with conventional storage by the U.S. Special Operations Department. .Currently there are no vaccines and medicines available for treating SEB-induced shock., however, therapeutic monoclonal antibodies become a preferred solution due to its high specificity and low toxicity. Several mouse-derived anti-SEB neutralizing monoclonal antibodies (mAbs) have been generated against SEB, but only partial inhibition was achieved. This fact demonstrates that a single monoclonal antibody is not sufficient to provide the protection against diseases induced by SEB. The combination of a pair of non-cross-reactive neutralizing anti-SEB monoclonal chimeric antibodies was reported to act synergistically against SEB in vitro and in vivo. Because each antibody has to be individually developed, tested for safety and efficacy, and then the whole thing has to be done also for the combination, it's really a doubling or even tripling of the effort required for developing a single antibody, which is already very costly.. In this study, we will construct a noval bispecific antibody 3E2-3C1 which can block the MHC Class II and TCR-binding sites on SEB surface simultaneously. We hope that 3E2-3C1 bispecific antibody was significantly more effective in inhibiting SEB induced lymphocyte activation in vitro and in vivo. It may constitute a better treatment strategy and provide a promising therapeutic agent for treatment of SEB intoxication.

金黄色葡萄球菌是医院院内感染常见的病原体之一，所产生的肠毒素是引起食物中毒的主要病因。而常规的抗生素治疗并不能清除体内的毒素，只有研制针对毒素的中和保护性抗体才能有效清除体内的毒素。SEB作为一类超抗原，直接与MHCII和TCR桥联结合而发挥作用，因此单一的单克隆抗体对SEB诱导的T淋巴细胞激活的抑制作用仅为50%左右。我们的前期研究表明：联合应用抗TCR及MHCII结合位点的鼠源单抗具有协同作用，因此我们设想构建表达能够同时结合SEB TCR和MHCⅡ功能域的双特异性中和性抗体，不但达到同时阻断SEB与TCR和MHCⅡ结合的目的，还可能造成其空间构象的变化，影响SEB的功能。本研究拟利用计算机辅助设计构建抗SEB双特异性抗体，检测双特异性抗体的特性和体内、外生物学功能，并探讨其中和保护作用的机制，为金黄色葡萄球菌的治疗提供新的有效的抗体制剂。

葡萄球菌肠毒素葡萄球菌肠毒素是由葡萄球菌分泌的细菌外毒素，目前已报道的有SEA、SEB、SEC1-3、SED、SEE、SEF、SEG等二十多种血清型，是细菌性食物中毒、化脓性感染、院内感染的重要毒素，而常规的抗生素治疗并不能清除体内的毒素，只有研制针对毒素的中和保护性抗体才能有效清除体内的毒素。SEB作为一类超抗原，直接与MHCII和TCR桥联结合而发挥作用，因此单一的单克隆抗体对SEB诱导的T淋巴细胞激活的抑制作用仅为50%左右。我们的前期研究表明,联合应用抗TCR及MHCII结合位点的鼠源单抗具有协同作用，在本研究中，通过计算机分子模拟构建了人源化的双可变区抗体3E1-3C1。 在ELISA竞争实验中，m3E2+m23C3(无关抗体)+DVD组对SEB抗原的竞争结合抑制为51%，m3C1+m23C3+DVD的竞争抑制为56%，而m3C1+m3E2+DVD的竞争抑制率为91%，表明我们所构建的DVD 3E2-3C1能同时竞争鼠源性单克隆抗体3C1 （TCRVβ的结合位点）和3E2 （MHC II结合位点）与SEB的结合，说明构建的DVD 3E2-3C1的具有同时结合SEB MHC II 和TCR结合功能域的能力。通过检测双特异性抗体的亲和力和体内、外生物学功能，发现DVD 3E2-3C1抗体的亲和力比人源化抗体h3E2、h3C1提高了近10倍,体内外的生物学功能明显优于单独使用人源化抗体和联合应用组。综上所述，我们所构建的双可变区抗体3E2-3C1能同时阻断SEB与TCR和MHCⅡ结合的目的，还可能造成其空间构象的变化，能大幅度提高抗体与SEB的亲和力，将大大提高单克隆抗体的中和保护作用，同时，作为单一的抗体分子，在疗效评价方面将比两个抗体分子更简便。为金黄色葡萄球菌的治疗提供新的有效的抗体制剂，还将为今后研制其它多表位的高活性抗毒素的基因工程抗体提供重要的基础理论支撑。