Engineering and Evaluation of Human L-Methionase for Cancer Therapy

人类 L-甲硫氨酸酶用于癌症治疗的工程和评估

基本信息

批准号：
8403663
负责人：
GEORGE Georgiou GEORGIOU
金额：
$ 40.31万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8403663
关键词：
Acute Adverse effects Alteplase Anabolism Anaphylaxis Animal Model Antibodies Apoptotic Biochemical Blood Circulation Buffers Cell Line Cells Cephalic Cessation of life Child Childhood Solid Neoplasm Clinical Clinical Trials Combination Drug Therapy Combined Modality Therapy Cystathionine Development Diagnosis Dietary Supplementation Disease Dose Drug Kinetics Effectiveness Engineering Enzyme Kinetics Enzymes Evaluation Exhibits Generations Glioblastoma Half-Life Homocysteine Homocystine Human Human Engineering Human Genome Hydrolysis Inhibitory Concentration 50 Injection of therapeutic agent Lead Left Liquid substance Lyase Malignant Neoplasms Malignant neoplasm of central nervous system Measures Mediating Metabolic Methionine Methylation Microtubule Depolymerization Mus Mutagenesis Neuroblastoma Non-Malignant Normal Cell Normal tissue morphology Nude Mice Patients Pharmaceutical Preparations Pharmacodynamics Physiological Plasma Polyamines Polymers Primates Process Property Prostate carcinoma Protein Biosynthesis Protein Engineering Proteins Pseudomonas Reaction Regimen Relapse Relative (related person)Reporting Serum Site Source Specificity Structure Substrate Specificity Techniques Therapeutic Toxic effect Variant Vincristine Work Xenograft Model Xenograft procedure advanced disease cancer therapy catalyst chemotherapy clinical practice clinically relevant combinatorial cytotoxic cytotoxicity design directed evolution efficacy evaluation enzyme structure established cell line high throughput screening immunogenic immunogenicity in vitro activity in vivo killings meetings mouse model mutant neoplastic neuroblastoma cell novel polypeptide pre-clinical public health relevance response tissue culture tumor tumor growth tumor xenograft

项目摘要

DESCRIPTION (provided by applicant): Neuroblastoma is the most common extra-cranial solid tumor of childhood with an appalling 30% cure rate in children with advanced disease. There is a clear need for new chemotherapeutics, as current drugs are only marginally effective at the high doses that result in toxic acute and grave long term side effects. The overall objective of this proposal is to employ modern techniques of protein engineering to develop a new generation of non-immunogenic and pharmacologically optimized enzymes for chemotherapy of neuroblastomas and other central nervous system (CNS) cancers through L-Methionine (L-Met) depletion. L-Met is required not only for protein synthesis but also as the precursor for methylation reductions and for the biosynthesis of polyamines. Tumors have a much greater requirement for L-Met than normal tissues and become apoptotic when its availability is restricted. i.v. administration of bacterial (Pseudomonas) methionine-g-lyase is able to mediated near complete depletion of L-Met in serum and has been shown to drastically inhibit tumor growth of neuroblastomas, glioblastomas and prostate carcinomas in mouse xeongrafts. Furthermore strong synergistic effects with microtubule depolymerization agents have been reported. Unfortunately, in clinical trials the bacterial enzyme was shown to have very poor pharmacological properties (t 1/2 in serum only 2 hrs) and was found to be highly immunogenic in primates eliciting severe adverse responses that resulted in anaphylactic shock and death. While the human genome does not encode any methionine lyase enzymes, in preliminary studies we deployed protein engineering strategies to generate potentially non-immunogenic variants of the human enzyme cystathionine-g-lyase that: (a) exhibit high L-Met degradation activity in vitro and in vivo, (b) display a lower IC50 for neuroblastoma cell lines than their bacterial counterparts and (c) are about 10-fold more stable in mice. Here we will employ structure guided mutagenesis and directed evolution strategies to: 1. Engineer catalytically optimized "human L-methioninases" i.e. cystathionine-g-lyase enzymes with very high activity for L-Met degradation, even better stability in serum and high selectivity. 2. Develop optimized formats of the "human L-methioninases" for prolonged persistence in circulation by either site-specific PEGylation or by fusion to long intrinsically disordered polypeptide sequences (XTEN) and determine their pharmacokinetic and pharmacodynamic properties. 3. Evaluate the efficacy of these enzymes in the mouse xenograft model of human neuroblastoma tumors formed using clinical cell lines established either in diagnosis or relapse. The utility of these enzymes will be investigated both as monotherapy and in combination therapy with vincristine.

描述（由申请人提供）：神经母细胞瘤是儿童时期最常见的颅外实体瘤，晚期疾病儿童的治愈率高达 30%，令人震惊。显然需要新的化疗药物，因为目前的药物在高剂量下效果有限，会导致急性毒性和严重的长期副作用。该提案的总体目标是采用现代蛋白质工程技术，开发新一代非免疫原性和药理学优化的酶，通过 L-蛋氨酸 (L-Met) 消耗，用于神经母细胞瘤和其他中枢神经系统 (CNS) 癌症的化疗。 L-Met 不仅是蛋白质合成所必需的，而且也是甲基化还原和多胺生物合成的前体。肿瘤对 L-Met 的需求量比正常组织大得多，当 L-Met 的可用性受到限制时，肿瘤就会发生凋亡。静脉注射细菌（假单胞菌）甲硫氨酸-g-裂解酶的施用能够介导血清中 L-Met 几乎完全耗尽，并且已被证明可以显着抑制小鼠异种移植物中神经母细胞瘤、胶质母细胞瘤和前列腺癌的肿瘤生长。此外，还报道了与微管解聚剂的强烈协同效应。不幸的是，在临床试验中，细菌酶被证明具有非常差的药理学特性（血清中的 t 1/2 仅 2 小时），并且发现在灵长类动物中具有高度免疫原性，引起严重的不良反应，导致过敏性休克和死亡。虽然人类基因组不编码任何蛋氨酸裂解酶，但在初步研究中，我们采用蛋白质工程策略来生成人类酶胱硫醚-g-裂解酶的潜在非免疫原性变体，其：(a) 在体外表现出高 L-Met 降解活性在体内，(b) 对神经母细胞瘤细胞系的 IC50 低于细菌细胞系，(c) 在小鼠体内的稳定性大约高 10 倍。在这里，我们将采用结构引导诱变和定向进化策略来： 1. 设计催化优化的“人 L-蛋氨酸酶”，即胱硫醚-g-裂解酶，具有非常高的 L-Met 降解活性，甚至在血清中具有更好的稳定性和高选择性。 2.开发“人L-蛋氨酸酶”的优化形式，通过位点特异性聚乙二醇化或通过与长的内在无序多肽序列(XTEN)融合来延长循环中的持久性，并确定其药代动力学和药效学特性。 3. 评估这些酶在人类神经母细胞瘤小鼠异种移植模型中的功效，该模型是使用诊断或复发时建立的临床细胞系形成的。这些酶的效用将作为单一疗法和与长春新碱的联合疗法进行研究。