Mammalian platform for identification of polyclonal antibody cancer therapies

用于鉴定多克隆抗体癌症疗法的哺乳动物平台

• 批准号: 8718729
• 负责人: Annalee W. Nguyen
• 金额: $ 5.78万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718729
• 关键词: Address; Affinity; Antibodies; Antibody Repertoire; Antibody Therapy; Apoptosis; Apoptotic; Automobile Driving; Avidity; B-Lymphocytes; Bacteria; Binding; Biological Markers; Breast Cancer Cell; Breast Cancer Treatment; Cancer Detection; Cancer cell line; Cell Culture Techniques; Cell Line; Cell surface; Cells; Chinese Hamster Ovary Cell; Crowding; Development; Disease; Drug Formulations; ERBB2 gene; Engineering; Environment; Epidermal Growth Factor Receptor; Episome; Epitopes; FDA approved; Fab Immunoglobulins; Flow Cytometry; Goals; Growth; Growth Factor Receptors; Human; Humira; Immune Targeting; Immunoglobulin Fragments; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; In Vitro; Individual; Induction of Apoptosis; Learning; Libraries; Ligands; Light; Malignant Neoplasms; Mammalian Cell; Methods; Monoclonal Antibodies; Mus; Oligonucleotides; Organism; Parents; Prevention; Process; Production; Proteins; Receptor Down-Regulation; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Roche brand of trastuzumab; Scheme; Sorting - Cell Movement; Spleen; Staging; Structure; Surface; System; Testing; Therapeutic antibodies; Time; Transmembrane Domain; Variant; Xenograft Model; Yeasts; antibody engineering; anticancer research; base; cancer cell; cancer therapy; career; clinical efficacy; design; design and construction; experience; improved; in vitro Assay; in vitro testing; malignant breast neoplasm; meetings; mutant; novel; polyclonal antibody; preference; prevent; protein expression; public health relevance; receptor; screening; skills; technology/technique; variable region gene

DESCRIPTION (provided by applicant): The developmental path for breast cancer antibody therapeutics follows three stages: (1) discovery of an antibody with high affinity for a given target through screening of murine B-cells or antibody fragments expressed on the surface of non-mammalian cells, (2) development, including engineering for increased affinity and conversion to a human-like IgG antibody, typically in prokaryotic or yeast cells, followed by (3) manufacturing, including expression in FDA-approved mammalian cell lines, purification and formulation. This slow and costly process results in a single monoclonal antibody against a previously known target, in spite of the fact that current research indicates synergistic pools of antibodies can be much more effective. Moreover, many antibodies fail in manufacturing since expression level and stability were not criteria used during the initial selection. In this proposa, these three steps are condensed into a streamlined process using CHO cells, the preferred production host. Novel selection schemes are designed to identify pools of antibodies based on function, induction of apoptosis in cancer cells, as opposed to just affinity. The initial target fr antibody development with this system is HER2, a receptor over-expressed in approximately 20% of breast cancer cases and capable of inducing apoptosis of cancer cells when bound to some therapeutic antibodies. First, a mammalian antigen-binding fragment (Fab) surface display system will be developed to permit cytometric sorting of CHO cells expressing functional anti-HER2 Fab from background cells expressing an irrelevant Fab. Second, mutagenic libraries will be created and screened to identify variants of a low affinity anti-HER2 Fab with increased function and affinity. Flow cytometric sorting will be used to select for high affinity binding to soluble HER2 targets, and, separately, to select Fab molecules that result in downstream anti-cancer effects such as apoptosis and growth factor receptor down-regulation in a breast cancer cell line. Finally, a mouse will be immunized with a breast cancer cell line over-expressing HER2 and EGFR, the murine antibody repertoire recovered and transferred to the Fab display system. The murine- derived Fab sequences will be subjected to error-prone PCR and in vitro somatic hypermutation prior to screening to identify antibodies triggering downstream in vitro anti-cancer effects, providing ample opportunity for discovery of synergistic antibodies. The new antibodies will be transferred to a soluble IgG expression system, biochemically characterized, and tested in vitro for prevention of breast cancer cell line growth. Top performing antibodies will be humanized and tested as both monoclonal and polyclonal therapies in in vitro assays and, ultimately, a murine xenograft model. The technology and techniques developed in this proposal will result in a generalizable method for the discovery of more potent therapeutic antibody mixtures, and the resulting antibody cocktails will offer new options for the treatment of breast cancer.

描述（由申请人提供）：乳腺癌抗体疗法的发展路径分为三个阶段：（1）通过筛选鼠B细胞或非B细胞表面表达的抗体片段，发现对给定靶点具有高亲和力的抗体。哺乳动物细胞，(2) 开发，包括工程设计以提高亲和力和转化为类人 IgG 抗体，通常在原核或酵母细胞中，然后 (3) 制造，包括在 FDA 批准的哺乳动物细胞系中表达、纯化和配制。尽管当前的研究表明抗体的协同池可以更有效，但这种缓慢且昂贵的过程会产生针对先前已知靶标的单一单克隆抗体。此外，由于表达水平和稳定性不是初始选择期间使用的标准，许多抗体在生产中失败。在此提案中，这三个步骤被浓缩为使用 CHO 细胞（首选生产宿主）的简化流程。新颖的选择方案旨在根据功能、诱导癌细胞凋亡来识别抗体库，而不仅仅是亲和力。该系统开发抗体的最初目标是 HER2，这是一种在大约 20% 的乳腺癌病例中过度表达的受体，当与某些治疗抗体结合时能够诱导癌细胞凋亡。首先，将开发哺乳动物抗原结合片段 (Fab) 表面展示系统，以允许对表达功能性抗 HER2 Fab 的 CHO 细胞与表达不相关 Fab 的背景细胞进行细胞分析。其次，将创建和筛选诱变文库，以鉴定功能和亲和力增强的低亲和力抗 HER2 Fab 的变体。流式细胞术分选将用于选择与可溶性 HER2 靶标的高亲和力结合，并单独选择可导致下游抗癌作用（例如乳腺癌细胞系中的细胞凋亡和生长因子受体下调）的 Fab 分子。最后，用过度表达 HER2 和 EGFR 的乳腺癌细胞系对小鼠进行免疫，恢复小鼠抗体库并转移至 Fab 展示系统。小鼠衍生的 Fab 序列将在筛选之前进行易错 PCR 和体外体细胞超突变，以鉴定触发下游体外抗癌作用的抗体，为发现协同抗体提供充足的机会。新抗体将被转移到可溶性 IgG 表达系统中，进行生化表征，并进行体外测试，以预防乳腺癌细胞系生长。表现最佳的抗体将被人源化，并在体外试验中作为单克隆和多克隆疗法进行测试，并最终在鼠异种移植模型中进行测试。该提案中开发的技术和技巧将导致发现更有效的治疗性抗体混合物的通用方法，并且由此产生的抗体混合物将为乳腺癌的治疗提供新的选择。