CORE--EXPRESSION AND PRODUCTION

Core C的支持功能，Expression和Production，有三个方面： 1)以瞬时表达新的生物工程抗体构建体， 稳定的哺乳动物表达系统，2）生产和纯化这些 用于项目2中计划的临床前研究的抗体构建体， 生物工程抗体和项目3，抗体螯合物和偶联物， 以及3）生产和纯化用于人体成像的临床级材料 以及本项目拟开展的I/II期治疗试验。 新的抗CEA生物工程抗体将在大肠杆菌中瞬时表达。 COS-1细胞系统 瞬时表达将允许快速的 新的生物工程构建体的生产和评价， 适当的折叠、分子大小、亲和力和亲合力。 如果初始 生化分析看起来很有希望，候选构建体将被 在Celltech谷氨酰胺合成酶（GS）哺乳动物表达中表达 系统 目前，cT84.66 GS18 Flex-ser微抗体在大肠杆菌中的表达不稳定。 GS系统在高产细胞系中显示出优异的结果 在固定瓶中表达10-50 ug/m1。 用于临床前研究的细胞培养上清液将通过离子色谱法纯化。 交换层析和抗独特型亲和柱。 抗体 构建体将在固定的T-烧瓶中生产，以提供1 mg批次的 用于体外表征和动物试验的纯化材料 药代动力学研究。 有限数量的位点特异性缀合 构建体，其显示用于进一步的蛋白质合成的所需标准。 结合研究，将以100 mg批次生产和纯化， 微型发酵罐生物反应器。 核心C将为三个生物工程选择高产细胞系 抗体构建体：cT84.66微抗体、T84.66双抗体和 人源化T84.66抗体。 生物工程抗体将在 一个最先进的中空纤维生物反应器 除了小说 在生物工程构建体中，核心C将产生4-5克嵌合T8.66 抗体并产生F（ab '）2片段用于继续人类治疗 审判 抗体将根据本发明中建立的方案进行纯化。 项目前期。 细胞系和纯化的抗体将满足 现行FDA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.