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

• 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
• 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

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）：神经母细胞瘤是最常见的儿童颅外实体瘤，患有晚期疾病的儿童的治愈率令人震惊。显然需要新的化学治疗药，因为目前的药物仅在高剂量的高剂量上略有有效，从而导致有毒的急性和严重的长期副作用。该提案的总体目的是利用蛋白质工程的现代技术来开发新一代的非免疫原性和药理学优化的酶，以通过L-Methionine（L-Met）deptletion进行神经细胞瘤和其他中枢神经系统（CNS）癌症的化学疗法。 L-MET不仅需要蛋白质合成，而且还需要作为甲基化还原和多胺生物合成的前体。肿瘤对L-MET的需求比正常组织要大得多，并且在限制其可用性时会凋亡。 I.V.细菌（假单胞菌）蛋氨酸-G-酯化酶的给药能够介导血清中L-MET的几乎完全消耗，并已显示出极大地抑制神经母细胞母细胞瘤，胶质母细胞母细胞瘤和前列腺癌的肿瘤生长。此外，已经报道了与微管解聚剂的强大协同作用。不幸的是，在临床试验中，细菌酶的药理特性非常差（血清中t 1/2仅2小时），并且发现在引起严重不良反应的灵长类动物中发现高度免疫原性，从而导致过敏性休克和死亡。虽然人类基因组没有编码任何甲基氨基酸性酶酶，但在初步研究中，我们部署了蛋白质工程策略，以产生人类酶胱硫硫氨酸 - 裂解酶的潜在非免疫原性变体，这些变异是：（a）比L-met在易于范围内的BACERAMA高l-met降解活性。 （c）在小鼠中稳定约10倍。在这里，我们将采用结构引导的诱变和定向的进化策略来：1。催化优化的“人类L-甲硫代蛋白酶”，即具有很高活性的L-MET降解活性，血清中更好的稳定性和高选择性的cystathionine-g-裂解酶。 2。开发“人类L-甲二世酶”的优化格式，以通过位点特异性的卵高或融合到长期固有无序多肽序列（XTEN）并确定其药物动力学和药物动力学特性。 3。评估这些酶在使用诊断或复发中建立的临床细胞系形成的人神经母细胞瘤肿瘤的小鼠异种移植模型中的功效。这些酶的实用性将作为单一疗法和vincristine组合疗法进行研究。