Mammalian platform for identification of polyclonal antibody cancer therapies

描述（由申请人提供）：乳腺癌抗体治疗的发展路径遵循三个阶段：（1）通过筛选鼠B细胞或在非哺乳动物细胞表面上表达的抗体片段，发现对给定靶具有高亲和力的抗体，（2）开发，包括用于增加亲和力的工程改造和转化为人样IgG抗体，通常在原核或酵母细胞中，随后（3）制造，包括在FDA批准的哺乳动物细胞系中表达、纯化和配制。这种缓慢且昂贵的过程导致针对先前已知的靶点的单一单克隆抗体，尽管目前的研究表明抗体的协同池可能更有效。此外，许多抗体在制造中失败，因为表达水平和稳定性不是初始选择期间使用的标准。在该提案中，这三个步骤被浓缩为使用CHO细胞（优选的生产宿主）的简化工艺。设计了新的选择方案，以基于功能、癌细胞凋亡的诱导来鉴定抗体库，而不仅仅是亲和力。使用该系统开发抗体的初始靶点是HER2，这是一种在约20%的乳腺癌病例中过表达的受体，当与一些治疗性抗体结合时能够诱导癌细胞凋亡。首先，将开发哺乳动物抗原结合片段（Fab）表面展示系统，以允许表达功能性抗HER2 Fab的CHO细胞与表达不相关Fab的背景细胞进行细胞计数分选。其次，将创建并筛选诱变文库，以鉴定功能和亲和力增加的低亲和力抗HER2 Fab变体。将使用流式细胞术分选来选择与可溶性HER2靶标的高亲和力结合，并分别选择导致下游抗癌作用的Fab分子，例如乳腺癌细胞系中的细胞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，回收鼠抗体库并转移至Fab展示系统。在筛选之前，对鼠源Fab序列进行易错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，并在体外进行测试，以预防乳腺癌细胞系的生长。表现最好的抗体将被人源化，并在体外测定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方法将导致发现更有效的治疗性抗体混合物的可推广方法，并且所产生的抗体混合物将为乳腺癌的治疗提供新的选择。

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.