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(申请人提供)：乳腺癌抗体疗法的发展道路经历了三个阶段：(1)通过筛选小鼠B细胞或非哺乳动物细胞表面表达的抗体片段，发现对特定靶点具有高亲和力的抗体，(2)开发包括提高亲和力并将其转化为类人抗体的工程，通常是在原核细胞或酵母细胞中，随后是(3)制造，包括在FDA批准的哺乳动物细胞系中表达，纯化和制备。尽管目前的研究表明，协同抗体库可能会更有效，但这一缓慢而昂贵的过程会产生针对先前已知靶点的单一单抗。此外，许多抗体的制造失败，因为表达水平和稳定性不是最初选择时使用的标准。在这个提议中，这三个步骤被浓缩成一个使用CHO细胞的简化过程，CHO细胞是首选的生产主机。新的选择方案旨在根据功能和诱导癌细胞凋亡来识别抗体库，而不仅仅是亲和力。该系统的最初目标是HER2，它是一种受体，在大约20%的乳腺癌病例中过度表达，当与一些治疗性抗体结合时，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，以便从表达无关Fab的背景细胞中分离表达功能性抗HER2 Fab的CHO细胞。其次，将建立和筛选诱变文库，以确定具有更高功能和亲和力的低亲和力抗HER2 Fab的变体。流式细胞术将用于选择与可溶性HER2靶标高亲和力结合的Fab分子，并分别选择导致乳腺癌细胞系中细胞凋亡和生长因子受体下调等下游抗癌作用的Fab分子。最后，用高表达HER2和EGFR的乳腺癌细胞株免疫小鼠，回收小鼠抗体库并转移到Fab展示系统中。鼠源性Fab序列在筛选前将经过容易出错的聚合酶链式反应和体外体细胞超突变，以识别触发体外抗癌作用的下游抗体，为发现协同抗体提供充足的机会。新的抗体将被转移到一个可溶的免疫球蛋白表达系统中，进行生化特征鉴定，并在体外进行测试，以防止乳腺癌细胞系的生长。表现最好的抗体将人源化，并在体外试验中作为单克隆和多克隆疗法进行测试，最终将作为小鼠异种移植模型进行测试。这项建议中开发的技术和技术将导致一种可推广的方法，用于发现更有效的治疗性抗体混合物，由此产生的抗体鸡尾酒将为乳腺癌的治疗提供新的选择。