AFFINITY ENGINEERING OF ANTIPOLYSACCHARIDE MONOCLONAL AN

许多病原菌都被外部多糖层包围 称为胶囊。 荚膜在细菌的毒力和 细菌，帮助他们逃避非特异性免疫反应， 宿主防御，因为抗荚膜抗体反应涉及 清除和保护。 以细菌莱万为模型 多糖，我们正在研究如何直接改变抗体蛋白 序列可用于影响抗多糖的亲合力增加 mAb. 这些亲合力工程抗体是由 抗体表达构建体的定向诱变和随后的 在合适的细胞底物中表达和抗体产生。 亩 已经制备了抗果聚糖单克隆的cDNA和κ链cDNA， 克隆到基于CMV启动子的表达载体中。 这些表达 载体已经在VH区突变以影响N-> D和N-> H 在位置53处改变。 这些表达载体目前正在 转染入仅产生杂交瘤细胞底物的κ链 用于产生突变的mAb。 工程化mAb将用于 研究单个氨基酸变化如何影响亲合力和特异性。 这些预测的变化也正在使用“看”软件进行建模 由美国国立卫生研究院DCRT分子建模中心在高功率 Silicon Graphics工作站 单个突变对 将确定抗体结合位点的构象。

Many pathogenic bacteria are surrounded by an outer polysaccharide layer referred to as the capsule. The capsule plays a role in both virulence of the bacteria, helping them evade the non-specific immune response, and in host defense, as anti-capsular antibody responses are involved with clearance and protection. Using bacterial levan as a model polysaccharide, we are studying how directed changes in antibody protein sequence can be used to affect avidity increases in anti-polysaccaride mAb. These avidity engineered antibodies are being produced by site directed mutagenesis of antibody expression constructs and subsequent expression and antibody production in an appropriate cell substrate. Mu and Kappa chain cDNAs of an anti-levan monoclonal have been produced and cloned into a CMV promotor based expression vector. These expression vectors have been mutated in the VH region to affect a N->D and a N->H change at position 53. These expression vectors are currently being transfected into a Kappa chain only producing hybdridoma cell substrate for production of the mutated mAb. The engineered mAb will be used to study how individual amino acid changes affect avidity and specificity. These predicted changes are also being modeled using the "Look" software provided by the Center for Molecular Modeling, DCRT, NIH on a high powered Silicon Graphics workstation. The effects of single mutations on the conformation of the antibody combining site will be determined.