PROTEIN EXPRESSION AND BISPECIFIC ANTIBODIES

蛋白质表达和双特异性抗体

• 批准号: 6161120
• 负责人: David M. Segal
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161120
• 关键词: antibody specificity; antitumor antibody; cytotoxic T lymphocyte; gene therapy; glycosylation; protein engineering; protein folding; protein structure function; virus infection mechanism

We have previously used bispecific antibodies (bsAb) to redirect cytotoxic cells against tumor and other target cells and to study triggering mechanisms. In a collaboration with GenVec, we are developing a new application for bsAbs, the selective targeting of cells for adenovirus infection. A bsAb with anti-CD3 and anti-FLAG activity induces the infection of human T cell with adenovirus that contains a genetically introduced FLAG peptide and expresses - galactosidase as a reporter gene. T cells are not infected without the bsAb, both CD4 and CD8 T cells are infected, and CD3- cells are not infected. Other bsAbs are being produced to see which types of molecules on cell surfaces can be used to induce bsAb dependent infections. BsAb directed infection could prove important for gene therapy applications. We have produced single chain (sc) bsAbs by concatenating two scFvs. These molecules are secreted as active protein by mammalian cells, or are produced by bacteria and refolded in vitro. We have found that inclusion of a glycosylation site on an anti-DNP scFv enhances its rate of secretion, without affecting its stability. More recently we have found that in the ER, the glycosylation site increases the rate of protein folding as measured by the appearance of anti-DNP binding activity. Preliminary studies, in collaboration with Kelly Kearse, showed that the glycosylated form of the scFv binds calnexin. We will use this system to see if calnexin aids the scFv in gaining anti-DNP activity. In our studies of in vitro protein folding we found that proteins that are normally secreted require special procedures for producing a native molecule. To see how a cytoplasmic protein would fold in vitro, we, in collaboration with Charles Zacharchuk, expressed the cytoplasmic portion of bcl-2 as inclusion bodies in bacteria. We found that after denaturation with guanidine and dialysis, the bcl-2 protein adopted a biologically active form that consisted of 2 non-covalently interacting regions separated by a long, exposed, protease-sensitive loop. The hydrodynamic properties of bcl-2 suggest that its structure is similar to the recently published X- ray structure of bcl-x. This first example suggests that cytoplasmic proteins adopt their native configuration more readily than secreted ones. Formerly Z01 CB 10028-01 EIB.

我们以前使用双特异性抗体（bsAb）重定向 细胞毒性细胞对肿瘤和其他靶细胞，并研究 触发机制。在与GenVec的合作中，我们正在开发 bsAbs的一个新应用，选择性靶向细胞， 腺病毒感染具有抗CD 3和抗FLAG活性的bsAb 诱导人T细胞感染腺病毒 基因引入FLAG肽并表达β-半乳糖苷酶作为一种 报告基因T细胞在没有bsAb的情况下不会被感染，CD 4和 CD 8 T细胞被感染，而CD 3-细胞未被感染。其他bsAbs 来观察细胞表面的哪种分子 用于诱导bsAb依赖性感染。BsAb定向感染 对基因治疗的应用很重要 我们通过连接两个scFv产生单链（sc）bsAb。 这些分子作为活性蛋白由哺乳动物细胞分泌，或 由细菌产生并在体外重折叠。我们发现 在抗DNP scFv上包含糖基化位点提高了其速率 不影响其稳定性。最近， 发现在ER中，糖基化位点增加了 通过抗DNP结合的出现测量的蛋白质折叠 活动与Kelly Kearse合作的初步研究， 显示scFv的糖基化形式结合钙连接蛋白。我们将 使用该系统来观察钙连接蛋白是否有助于scFv获得抗DNP 活动 在我们对体外蛋白质折叠的研究中，我们发现 通常需要特殊的程序来产生天然的 分子。为了观察细胞质蛋白质在体外是如何折叠的， 与Charles Zacharchuk合作，表达了细胞质部分， bcl-2在细菌中以包涵体形式存在。我们发现， 经胍变性和透析后，bcl-2蛋白呈变性， 生物活性形式，由2个非共价相互作用 由一个长的暴露的蛋白酶敏感环隔开的区域。的 bcl-2的水动力学性质表明，其结构类似于 最近发表的bcl-x的X射线结构。该第一示例 表明细胞质蛋白更多地采用它们的天然构型， 而不是隐藏的。以前称为Z 01 CB 10028-01 EIB。