Mammalian platform for identification of polyclonal antibody cancer therapies

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)通过筛选小鼠b细胞或在非哺乳动物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；(2)开发，包括在原核细胞或酵母细胞中提高亲和力并转化为类人IgG抗体的工程；随后(3)制造，包括在fda批准的哺乳动物细胞系中表达、纯化和配方。尽管目前的研究表明协同抗体池可能更有效，但这个缓慢而昂贵的过程会产生针对先前已知靶点的单一单克隆抗体。此外，许多抗体在生产中失败，因为在最初的选择中没有使用表达水平和稳定性的标准。在这个提议中，这三个步骤被浓缩成一个流线型的过程，使用CHO细胞，首选的生产宿主。新的选择方案被设计来识别基于功能的抗体池，诱导癌细胞凋亡，而不仅仅是亲和力。该系统抗体开发的初始目标是HER2，这是一种在约20%的乳腺癌病例中过度表达的受体，当与一些治疗性抗体结合时，能够诱导癌细胞凋亡。首先，将开发一种哺乳动物抗原结合片段（Fab）表面显示系统，以便从表达不相关Fab的背景细胞中对表达功能性抗her2 Fab的CHO细胞进行细胞分选。其次，将创建和筛选诱变文库，以鉴定具有增强功能和亲和力的低亲和力抗her2 Fab的变体。流式细胞分选将用于选择与可溶性HER2靶点高亲和力结合的Fab分子，并单独选择在乳腺癌细胞系中产生下游抗癌作用的Fab分子，如凋亡和生长因子受体下调。最后，用过表达HER2和EGFR的乳腺癌细胞系免疫小鼠，小鼠抗体库恢复并转移到Fab显示系统。小鼠来源的Fab序列将在筛选之前进行易出错的PCR和体外体细胞超突变，以确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。新的抗体将被转移到可溶性IgG表达系统中，进行生物化学表征，并在体外进行预防乳腺癌细胞系生长的测试。表现最好的抗体将被人源化，并在体外试验中作为单克隆和多克隆治疗进行测试，并最终在小鼠异种移植模型中进行测试。本提案中开发的技术和技术将为发现更有效的治疗性抗体混合物提供一种可推广的方法，由此产生的抗体鸡尾酒将为治疗乳腺癌提供新的选择。

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.