Engineering Inhibitory Antibodies to Ectoenzymes for Cancer Treatment

描述(申请人提供)：抗体已经成为诊断和治疗中越来越重要的试剂，对结合到蛋白质靶标的给定位置的新型设计分子的需求也越来越大。现有的方法允许产生针对给定线性表位的抗体。然而，折叠蛋白表面的大多数位置都是构象表位，目前还没有获得针对预先指定的构象表位的抗体的技术手段。这一限制是一个重要的问题，因为这阻碍了基于抗体的新疗法的发展。我们提出了一种新的方法来设计结合到折叠蛋白的预先指定的表位的抗体。我们希望将这项技术应用于产生胞外酶的抑制性抗体，胞外酶在肿瘤进展和转移中起主要作用。特别是，我们已经证明了细胞表面的胞外核糖核酸酶CD39水解胞外核苷酸产生腺苷，腺苷强烈抑制抗肿瘤免疫并促进血管生成。用小分子化合物多金属氧酸盐-1抑制CD39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技术，用于设计与折叠蛋白的给定表位结合的抗体，并例如生产一种有效的、选择性的小鼠CD39抗体抑制物。我们假设CD39和其他酶的抑制性抗体可以通过靶向酶的活性部位从共同的前体抗荧光素抗体中获得。我们将对这一假设进行如下检验。目的1：通过靶向CD39的活性部位，建立TH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，在CD39的活性部位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，其中酶活性完全被抗荧光素抗体抑制，表明抗体阻断活性部位的最佳位置。目的2：获得不依赖于荧光素标记的抑制性抗荧光素抗体与CD39的结合互补性。我们将使用一种传统的“亲和力成熟”方法，通过随机化抗体的互补性决定区域和选择改善结合。我们预计，选择结合将保留抑制功能，并产生针对小鼠CD39的抑制性抗体。我们预计会产生以下积极影响：首先，针对小鼠CD39的抑制性抗体将使我们能够在后续的动物研究中评估靶向CD39在癌症中的全部治疗潜力。其次，这项衍生技术将使针对人类CD39和其他与癌症进展有关的关键胞外酶的抑制性抗体能够进行合理的工程设计。此外，开发的技术将满足研究和诊断方面的需求，并从根本上推动治疗性抗体工程领域的发展。 公共卫生相关性：癌症进展严重依赖于细胞表面胞外核苷酸酶的活性。我们建议开发一种新的技术来生产这些酶的抗体抑制剂用于癌症治疗。

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.