Engineering Inhibitory Antibodies to Ectoenzymes for Cancer Treatment

用于癌症治疗的胞外酶工程抑制性抗体

• 批准号: 8309768
• 负责人: SIMON C. ROBSON
• 金额: $ 22.71万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8309768
• 关键词: Active Sites; Adenosine; Affinity; Animals; Antibodies; Antibody Binding Sites; Antibody Formation; Antibody Therapy; Antigens; Binding; Blocking Antibodies; Cell surface; Complementarity Determining Regions; Cysteine; Development; Diagnostic; Diagnostics Research; Engineering; Enzymes; Epitopes; Fluorescein; Goals; Human; Immunosuppression; Label; Libraries; Malignant Neoplasms; Membrane; Methodology; Methods; Mus; Mutagenesis; Neoplasm Metastasis; Nucleotides; Outcome; Phage Display; Play; Positioning Attribute; Proteins; Purinoceptor; Randomized; Role; Series; Signal Transduction; Site; Site-Directed Mutagenesis; Specific qualifier value; Surface; Technology; Testing; Therapeutic; Therapeutic Effect; Therapeutic antibodies; Toxic effect; Tumor Immunity; Variant; Work; angiogenesis; antibody engineering; antibody inhibitor; base; cancer therapy; extracellular; improved; meetings; mutant; new technology; novel; novel strategies; protein folding; research study; small molecule; three dimensional structure; tumor growth; tumor progression

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.

描述（由申请人提供）：抗体已成为诊断和治疗中越来越重要的试剂，并且对结合蛋白质靶标的给定位点的新型设计分子的需求日益增长。现有方法允许产生针对给定线性表位的抗体。然而，折叠蛋白表面上的大多数位点是构象表位，并且目前没有用于获得针对预先指定的构象表位的抗体的技术手段。这种限制是一个重要的问题，因为这阻碍了新的基于抗体的治疗剂的开发。我们提出了一种新的方法，工程抗体结合到一个预先指定的表位的折叠蛋白质。我们希望应用该技术来生产针对在肿瘤进展和转移中起主要作用的胞外酶的抑制性抗体。特别是，我们已经表明，细胞表面外核苷酸酶CD 39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。用小分子化合物聚氧乙烯酯-1抑制CD 39可显著抑制肿瘤生长，但不良的选择性和毒性限制了治疗效果。本申请的目的是开发用于工程化抗体的通用技术，所述抗体结合折叠蛋白的给定表位，并且作为实例，产生小鼠CD 39的有效和选择性抗体抑制剂。我们假设，CD 39和其他酶的抑制性抗体可以通过靶向酶的活性位点从共同的前体抗荧光素抗体工程化。我们将按如下方式检验这一假设。目的1：以CD 39的活性位点为靶点，建立抗荧光素抗体的抑制功能。基于CD 39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CD 39的活性位点附近创建人工抗体结合位点。然后，我们将鉴定其中酶活性被抗荧光素抗体完全抑制的CD 39突变体，这表明抗体用于阻断活性位点的最佳位置。目的2：在抑制性抗荧光素抗体和CD 39之间产生不依赖于荧光素标记的结合互补性。我们将使用“亲和力成熟”的传统方法，通过随机化抗体的互补决定区和选择以改善结合。我们预期结合的选择将保留抑制功能并产生针对小鼠CD 39的抑制性抗体。我们预期以下积极影响：首先，小鼠CD 39的抑制性抗体将使我们能够在随后的动物研究中评估靶向CD 39在癌症中的全部治疗潜力。第二，衍生技术将使合理的工程抑制抗体的人CD 39和其他关键胞外酶参与癌症的进展。此外，所开发的技术将满足研究、诊断的需求，并从根本上推进治疗性抗体工程领域。 公共卫生相关性：癌症进展严重依赖于细胞表面外核苷酸酶的活性。我们建议开发一种新的技术来生产用于癌症治疗的这些酶的抗体抑制剂。