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

描述（由申请人提供）：单克隆抗体是破译基因或蛋白质功能的必要试剂，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靶向聚糖用于这些目的一直不太成功。与蛋白质或核酸相比，聚糖含有较少的疏水功能，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支链寡糖的微妙变化，这些变化不能用常规抗体检测到。因此，很难获得能够完全了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）有关；因此，鉴定特异性和高亲和力聚糖抗体的通用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。我们建议利用新的抗体噬菌体展示技术来开发鉴定这些试剂的方法。最近，从噬菌体文库中选择几乎任何蛋白质抗原的特异性和高亲和力抗体已经成为可能，这些噬菌体文库具有由合成DNA编码的定制多样性元素（“合成抗体”）。这种创新的技术平台避免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2G12的独特结构支架作为我们设计的模板，2G12是一种靶向HIV-1糖蛋白gp120上寡核苷酸的抗体。2G12 IgG的两个重链可变结构域交换位置，形成一个扩展的识别表面，每个IgG分子含有四个低聚糖结合位点。我们已经开发出了一种噬菌体载体，它允许2G12支架的功能展示。我们将准备合成2G12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，我们将利用从这些研究中获得的信息来鉴定具有改变肿瘤相关聚糖靶点特异性谱的新的2G12变体。这一创新策略将产生新的癌症抗体，加速癌症研究，为开发新的癌症治疗和诊断方法铺平道路。

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.