Engineering Inhibitory Antibodies to Ectoenzymes for Cancer Treatment

描述（由申请人提供）：抗体在诊断和治疗中已成为越来越重要的药物，并且对结合蛋白质靶点的特定位点的新型设计分子的需求日益增长。现有的方法允许生产针对给定线性表位的抗体。然而，折叠蛋白表面的大多数位点是构象表位，目前还没有技术手段来获得预先指定的构象表位的抗体。这种限制是一个重要的问题，因为它阻碍了基于抗体的新型治疗方法的发展。我们提出了一种结合折叠蛋白的预先指定表位的工程抗体的新方法。我们希望将这项技术应用于生产在肿瘤进展和转移中起主要作用的外切酶的抑制性抗体。特别是，我们已经证明细胞表面外核苷酸酶CD39水解细胞外核苷酸产生腺苷，这强烈抑制抗肿瘤免疫和促进血管生成。小分子化合物多金属酸氧酯-1对CD39的抑制作用可显著抑制肿瘤生长，但较差的选择性和毒性限制了治疗效果。本申请的目的是开发一种通用的工程抗体技术，结合折叠蛋白的给定表位，并作为一个例子，产生一种有效的和选择性的小鼠CD39抗体抑制剂。我们假设CD39和其他酶的抑制性抗体可以由一个共同的前体抗荧光素抗体通过靶向酶的活性位点来设计。我们将以如下方式检验这一假设。目的1：通过靶向CD39的活性位点，建立抗荧光素抗体的抑制功能。基于CD39的三维结构，我们将通过诱变引入半胱氨酸残基，并用荧光素标记这些残基，在CD39的活性位点附近创建人工抗体结合位点。然后，我们将鉴定一个CD39突变体，其中酶活性被抗荧光素抗体完全抑制，表明抗体阻断活性位点的最佳位置。目的2：产生不依赖荧光素标签的抑制抗荧光素抗体与CD39之间的结合互补。我们将使用传统的“亲和成熟”方法，通过随机确定互补区域和选择改善结合的抗体。我们期望选择结合将保留抑制功能并产生小鼠CD39的抑制性抗体。我们预计以下积极影响：首先，小鼠CD39的抑制抗体将使我们能够在随后的动物研究中评估靶向CD39治疗癌症的全部治疗潜力。其次，该衍生技术将使针对人类CD39和其他与癌症进展有关的关键外酶的抑制抗体的合理工程成为可能。此外，开发的技术将满足研究、诊断的需求，并从根本上推动治疗性抗体工程领域的发展。

DESCRIPTION (provided by applicant): Antibodies have become increasingly important agents in diagnostics and therapy, and there are growing demands for novel designer molecules that bind to a given site of a protein target. The existing methods allow production of antibodies against a given linear epitope. However, the majority of sites on the surface of a folded protein are conformational epitopes, and currently there are no technical means for obtaining antibodies to a pre-specified conformational epitope. This limitation is an important problem as this impedes development of novel antibody-based therapeutics. We propose a novel approach for engineering antibodies that bind to a pre-specified epitope of a folded protein. We wish to apply this technology for producing inhibitory antibodies to ectoenzymes that play a principal role in tumor progression and metastasis. In particular, we have shown that the cell surface ectonucleotidase CD39 hydrolyzes extracellular nucleotides to produce adenosine, which strongly suppresses anti-tumor immunity and promotes angiogenesis. Inhibition of CD39 with a small-molecule compound, polyoxometalate-1, significantly inhibits tumor growth, but poor selectivity and toxicity limit the therapeutic effects. The objective of thi application is to develop a universal technology for engineering antibodies that bind to a given epitope of a folded protein, and as an example, produce a potent and selective antibody inhibitor of mouse CD39. We hypothesize that inhibitory antibodies to CD39 and other enzymes can be engineered from a common precursor anti-fluorescein antibody by targeting active sites of enzymes. We will test this hypothesis as follows. Aim 1: Establish the inhibitory function of th anti-fluorescein antibody by targeting the active site of CD39. Based on the 3D structure of CD39, we will create artificial antibody-binding sites near the active site of CD39 by introducing cysteine residues via mutagenesis and labeling these with fluorescein. We will then identify a CD39 mutant where enzymatic activity is completely inhibited by the anti-fluorescein antibody, indicating the optimal position of the antibody for blocking the active site. Aim 2: Generate binding complementarity between the inhibitory anti-fluorescein antibody and CD39 independent of fluorescein label. We will use a traditional approach of "affinity maturation" by randomization of complementarity determining regions of the antibody and selection for improved binding. We expect that selection for binding will preserve inhibitory functions and produce the inhibitory antibody to mouse CD39. We anticipate the following positive impacts: First, the inhibitory antibody to mouse CD39 will allow us to evaluate in subsequent animal studies the full therapeutic potential of targeting CD39 in cancer. Second, the derived technology will enable rational engineering of inhibitory antibodies to human CD39 and other key ectoenzymes implicated in cancer progression. In addition, the developed technology will meet needs in research, diagnostics and fundamentally advance the field of therapeutic antibody engineering. PUBLIC HEALTH RELEVANCE: Cancer progression critically depends on activity of cell surface ectonucleotidases. We propose to develop a novel technology to produce antibody inhibitors of these enzymes for cancer treatment.