Methods to Identify High-Affinity Antibodies that Target Tumor-Associated Glycans

描述（由申请人提供）：单克隆抗体是解读基因或蛋白质功能的基本试剂，是治疗和诊断试剂的丰富来源。然而，使用抗碳水化合物抗体来靶向聚糖用于这些目的并不太成功。聚糖含有比蛋白质或核酸更少的疏水功能，因此单个聚糖-抗体相互作用相对较弱。由聚糖编码的信息通常涉及无法用常规抗体检测的分支寡糖的细微变化。因此，很难获得能够完全理解生物聚糖功能的试剂。细胞表面聚糖组成的变化与癌症（和其他疾病状态）相关;因此，鉴定特异性和高亲和力聚糖抗体的一般方法将极大地促进癌症研究并为癌症治疗和诊断提供新的途径。我们建议开发方法，以确定这种试剂使用新的抗体噬菌体展示技术。最近已经可以从携带由合成DNA编码的定制多样性元件的噬菌体文库中选择针对几乎任何蛋白质抗原的特异性和高亲和力抗体（“合成抗体”）。这种创新的技术平台消除了对动物免疫的要求，从而规避了传统杂交瘤方法的许多局限性。我们将使用2G 12的独特结构支架作为我们设计的模板，2G 12是一种靶向HIV-1糖蛋白gp 120上的寡甘露糖的抗体。2G 12 IgG的两个重链可变结构域交换位置以产生每个IgG分子含有四个寡甘露糖结合位点的延伸识别表面。我们开发了一种噬菌体载体，可以功能性展示2G 12支架。我们将制备合成的2G 12抗体文库，以确定该支架中高亲和力聚糖识别的最低物理化学要求。接下来，我们将使用从这些研究中获得的信息来鉴定对肿瘤相关聚糖靶点具有改变的特异性谱的新型2G 12变体。这一创新策略将产生新型癌症抗体，加速癌症研究，并为开发新的癌症治疗和诊断方法铺平道路。 公共卫生相关性：细胞表面寡糖（“聚糖”）在癌症和许多其他疾病状态中发挥关键作用，但聚糖的生物学功能知之甚少。我们的目标是开发策略，以确定聚糖靶向抗体作为研究试剂，诊断，或治疗。我们的方法采用了新的抗体分离方法，规避了传统抗体技术的许多限制，因此将产生具有增强特性的抗体用于生物医学应用。

DESCRIPTION (provided by applicant): Monoclonal antibodies are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, the use of anticarbohydrate antibodies to target glycans for these purposes has been less successful. Glycans contain less hydrophobic functionality than do proteins or nucleic acids, thus individual glycan-antibody interactions are relatively weak. Information encoded by glycans often involves subtle variations of branched oligosaccharides that cannot be detected with conventional antibodies. It has therefore been difficult to obtain reagents that enable a complete understanding of biological glycan function. Changes in cell surface glycan composition are associated with cancer (and other disease states); therefore, general methods to identify specific and high- affinity glycan antibodies would greatly facilitate cancer research and provide new avenues for cancer therapies and diagnostics. We propose to develop methods to identify such reagents using novel antibody phage display technologies. It has recently become possible to select specific and high affinity antibodies for virtually any protein antigen from phage libraries that bear tailored diversity elements encoded by synthetic DNA ('synthetic antibodies'). This innovative technology platform obviates the requirement for animal immunization, thereby circumventing many limitations of traditional hybridoma methods. We will use the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for our design. The two heavy chain variable domains of 2G12 IgG exchange positions to create an extended recognition surface containing four oligomannose binding sites per IgG molecule. We have developed a phage vector that allows the functional display of the 2G12 scaffold. We will prepare synthetic 2G12 antibody libraries to determine minimal physicochemical requirements for high affinity glycan recognition in this scaffold. Next, we will use the information gained from these studies to identify novel 2G12 variants with altered specificity profiles for tumor-associated glycan targets. This innovative strategy will result in novel cancer antibodies that accelerate cancer research and pave the way for development of new cancer therapies and diagnostics. PUBLIC HEALTH RELEVANCE: Cell-surface oligosaccharides ('glycans') play critical roles in cancer and many other disease states, but the biological functions of glycans are poorly understood. Our goal is to develop strategies to identify glycan-targeting antibodies for use as research reagents, diagnostics, or therapeutics. Our approach employs novel methods for antibody isolation that circumvent many limitations of conventional antibody technologies, and will therefore yield antibodies with enhanced properties for biomedical applications.