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

基本信息

批准号：
8451262
负责人：
SIMON C. ROBSON
金额：
$ 17.79万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2015-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8451262
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

项目摘要

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.

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，并且对结合到蛋白质靶标的给定位点的新型设计分子的需求不断增长。现有的方法允许产生针对给定线性表位的抗体。然而，折叠蛋白表面的大部分位点都是构象表位，目前还没有技术手段可以获得针对预先指定的构象表位的抗体。这种限制是一个重要的问题，因为这阻碍了新型抗体疗法的开发。我们提出了一种新的方法来工程化与折叠蛋白的预先指定的表位结合的抗体。我们希望应用这项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，我们发现细胞表面核酸外切酶 CD39 会水解细胞外核苷酸产生腺苷，从而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可显着抑制肿瘤生长，但选择性差和毒性限制了治疗效果。该应用的目的是开发一种通用技术，用于工程化抗体，使其与折叠蛋白的给定表位结合，例如，生产小鼠 CD39 的有效且选择性的抗体抑制剂。我们假设可以通过靶向酶的活性位点，从常见的前体抗荧光素抗体中设计出针对 CD39 和其他酶的抑制性抗体。我们将按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3D结构，我们将通过诱变引入半胱氨酸残基并用荧光素标记它们，在CD39的活性位点附近创建人工抗体结合位点。然后，我们将鉴定出一种 CD39 突变体，其中酶活性被抗荧光素抗体完全抑制，表明该抗体用于阻断活性位点的最佳位置。目标 2：在抑制性抗荧光素抗体和 CD39 之间产生不依赖于荧光素标记的结合互补性。我们将使用传统的“亲和力成熟”方法，通过随机化抗体的互补性决定区域并选择改进的结合。我们预计结合选择将保留抑制功能并产生针对小鼠 CD39 的抑制性抗体。我们预计将产生以下积极影响：首先，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，衍生技术将能够合理设计针对人类 CD39 和其他与癌症进展有关的关键胞外酶的抑制性抗体。此外，所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。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity.

DOI：
10.3390/ijms21175998
发表时间：
2020-08-20
期刊：
International journal of molecular sciences
影响因子：
5.6
作者：
Feldbrügge L;Splith K;Kämmerer I;Richter S;Riddermann A;Ortiz Galindo SA;Krenzien F;Müller T;Csizmadia E;Pratschke J;Robson SC;Schmelzle M
通讯作者：
Schmelzle M

NTPDase2 and the P2Y1 receptor are not required for mammalian eye formation.

NTPDase2 和 P2Y1 受体不是哺乳动物眼睛形成所必需的。

DOI：
10.1007/s11302-014-9440-5
发表时间：
2015
期刊：
Purinergic signalling
影响因子：
3.5
作者：
Gampe,Kristine;Haverkamp,Silke;Robson,SimonC;Gachet,Christian;Hüser,Laura;Acker-Palmer,Amparo;Zimmermann,Herbert
通讯作者：
Zimmermann,Herbert

Hyperthermia and associated changes in membrane fluidity potentiate P2X7 activation to promote tumor cell death.

DOI：
10.18632/oncotarget.18595
发表时间：
2017-09-15
期刊：
Oncotarget
影响因子：
0
作者：
de Andrade Mello P;Bian S;Savio LEB;Zhang H;Zhang J;Junger W;Wink MR;Lenz G;Buffon A;Wu Y;Robson SC
通讯作者：
Robson SC

Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer.

DOI：
10.1016/j.jim.2017.04.003
发表时间：
2017-08
期刊：
Journal of immunological methods
影响因子：
2.2
作者：
Zhang H;Chan LL;Rice W;Kassam N;Longhi MS;Zhao H;Robson SC;Gao W;Wu Y
通讯作者：
Wu Y

Utility of the dual-specificity protein kinase TTK as a therapeutic target for intrahepatic spread of liver cancer.

DOI：
10.1038/srep33121
发表时间：
2016-09-13
期刊：
Scientific reports
影响因子：
4.6
作者：
Miao R;Wu Y;Zhang H;Zhou H;Sun X;Csizmadia E;He L;Zhao Y;Jiang C;Miksad RA;Ghaziani T;Robson SC;Zhao H
通讯作者：
Zhao H