PROTEIN EXPRESSION AND BISPECIFIC ANTIBODIES

我们以前曾使用双特异性抗体(BsAb)重定向 细胞毒细胞对肿瘤等靶细胞的杀伤作用及研究 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。一种具有抗CD3和抗FLAG活性的单抗 用腺病毒感染人T细胞 基因导入FLAG多肽并表达半乳糖苷酶 记者基恩。T细胞在没有bsAb的情况下不会感染，无论是CD4还是 CD8 T细胞被感染，CD3-细胞不被感染。其他bsAbbs 是为了看看细胞表面哪些类型的分子可以 用于诱导依赖于bsAb的感染。双链抗体定向感染 可能会被证明对基因治疗的应用很重要。 我们通过串联两个单链抗体制备了单链抗体。 这些分子由哺乳动物细胞以活性蛋白质的形式分泌，或者 是由细菌产生的，并在体外折叠。我们发现， 在抗DNP单链抗体上加入糖基化位点可提高其转化率 在不影响其稳定性的情况下，增加分泌物的含量。最近，我们有了 发现在内质网中，糖基化位点增加了 通过出现抗DNP结合来测量蛋白质折叠 活动。与凯利·科尔斯合作进行的初步研究， 结果表明，糖基化形式的ScFv与Calnexin结合。我们会 使用此系统查看calnexin是否有助于单链抗体获得抗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才能产生本地化的 分子。为了观察细胞质蛋白质在体外如何折叠，我们在 与查尔斯·扎克丘克的合作，表达了细胞质部分 在细菌中以包涵体的形式表达bcl2。我们发现，在 经DNA变性和透析后，bcl-2蛋白采用 由两个非共价相互作用组成的生物活性形式 区域之间有一个长的、暴露的、对蛋白水解酶敏感的环。这个 BCL-2的流体力学性质表明其结构相似 到最近发表的BCL-x的X射线结构。第一个示例 表明细胞质蛋白更多地采用它们的天然构型 而不是偷偷摸摸的人。原Z01 CB 10028-01 EIB。

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.