CORE--EXPRESSION AND PRODUCTION

核心C的支持功能，表达和生产，有三个方面： 1)表达新型生物工程抗体载体 稳定的哺乳动物表达系统，2)生产和纯化这些 在项目2中计划的临床前研究的抗体构建， 生物工程抗体和项目3，抗体螯合物和结合物， 3)临床级人体影像材料的制备和提纯 以及本项目建议的I/II期治疗试验。 新型抗CEA生物工程抗体将瞬时表达于 基于COS-1蜂窝的系统。瞬时表达将允许快速 新型生物工程载体的生产与评价 适当的折叠，分子大小，亲和力和亲和力。如果是初始的 生化分析看起来很有希望，候选结构将是 谷氨酰胺合成酶(GS)在哺乳动物中的表达 系统。目前，cT84.66 GS18 Flex-Ser微体在 GS系统在高产细胞系上表现出了很好的效果 在固定瓶中表达10-50ug/m1。 用于临床前研究的细胞培养上清液将通过离子提纯 交换层析和抗独特型亲和柱。抗体 构建物将在固定的T烧瓶中生产，以提供1毫克批次的 体外鉴定和动物用的纯化材料 药代动力学研究。有限数量的特定位点结合 构建，它显示了进一步蛋白质的所需标准 接合作用研究，将在一年内以100毫克批量生产和提纯 微型发酵罐生物反应器。 Core C将为三种生物工程选择高产细胞系 抗体结构：cT84.66微型体、T84.66 diabody和a 人源化T84.66抗体。生物工程抗体将在 一种最先进的中空纤维生物反应器。除了小说之外 生物工程构建，核心C将产生4-5克嵌合T8.66 产生抗体并产生F(ab‘)2片段，用于继续人类治疗 审判。抗体将根据 上一个项目期。细胞株和纯化的抗体将会相遇 美国食品和药物管理局现行指南中规定的规格。

The support function of Core C, Expression and Production, are three fold: 1) to express novel bioengineered antibody constructs in transient and stable mammalian expression systems, 2) to produce and purify these antibody constructs for preclinical studies planned in Project 2, Bioengineered Antibodies, and Project 3, Antibody Chelates and Conjugates, and 3) to produce and purify clinical grade material for the human imaging and Phase I/II therapy trials proposed in this Project. Novel anti-CEA bioengineered antibodies will be transiently expressed in a COS-1 cell based system. Transient expression will allow the rapid production and evaluation of novel bioengineered constructs for their proper folding, molecular size, affinity and avidity. If initial biochemical analysis looks promising, candidate constructs will be expressed in the Celltech glutamine synthetase (GS) mammalian expression system. Currently, expression of the cT84.66 GS18 Flex-ser minibody in the GS system has shown excellent results with high producing cell lines expressing 10-50 ug/m1 in stationary flasks. Cell culture supernatant for preclinical studies will be purified by ion exchange chromatography and an anti-idiotype affinity column. Antibody constructs will be produced in stationary T-flasks to provide 1 mg lots of purified material for in vitro characterization and animal pharmacokinetics studies. A limited number of site specific conjugation constructs, which show the desired criteria for further protein conjugation studies, will be produced and purified in 100 mg lots in a minifermenter bioreactor. Core C will select high-producing cell lines for three bioengineered antibody constructs: the cT84.66 minibody, the T84.66 diabody and a humanized T84.66 antibody. Bioengineered antibodies will be produced in a state-of-the-art hollow-fiber bioreactor. In addition to the novel bioengineered constructs, Core C will produce 4-5 grams of chimeric T8.66 antibody and generate F(ab')2 fragments for continuing human therapy trials. Antibody will be purified under protocols established in the previous Project period. The cell lines and purified antibody will meet the specifications set forth in the current FDA guidelines.