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: 8023816
• 负责人: GEORGE Georgiou GEORGIOU
• 金额: $ 44.25万
• 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/8023816
• 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; 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. PUBLIC HEALTH RELEVANCE: 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. We are developing a drug engineered from a human enzyme that can kill tumors as a single agent by attacking a key metabolic point of neuroblastoma cells and is showing fantastic promise when used in combination with low non-toxic doses of current chemotherapeutics.

描述(申请人提供)：神经母细胞瘤是最常见的儿童颅外实体瘤，在患有晚期疾病的儿童中，治愈率高达惊人的30%。显然需要新的化疗药物，因为目前的药物在导致毒性、急性和严重的长期副作用的大剂量下只有轻微的效果。这项建议的总体目标是利用现代蛋白质工程技术开发新一代非免疫原性和药物优化的酶，通过L-蛋氨酸(L-蛋氨酸)耗竭用于神经母细胞瘤和其他中枢神经系统癌症的化疗。L-蛋氨酸不仅是蛋白质合成所必需的，也是甲基化还原和多胺生物合成的前体。肿瘤对L-蛋氨酸的需求比正常组织高得多，当可获得性受到限制时，肿瘤就会发生凋亡。静脉注射。细菌(假单胞菌)蛋氨酸g-裂解酶能使血清中L-蛋氨酸接近完全耗竭，并能显著抑制小鼠神经母细胞瘤、胶质母细胞瘤和前列腺癌移植瘤的生长。此外，有报道称微管解聚剂具有很强的协同效应。不幸的是，在临床试验中，这种细菌酶的药理性质很差(血清中的t1/2只有2小时)，并且在灵长类动物中被发现具有高度的免疫原性，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产生人类胱硫氨酸裂解酶的潜在非免疫原性变体：(A)在体外和体内显示出高的L-蛋氨酸降解活性，(B)对神经母细胞瘤细胞株显示比其细菌更低的IC50，(C)在小鼠体内表现出约10倍的稳定性。在这里，我们将采用结构导向突变和定向进化策略：1.工程优化的人L蛋氨酸酶，即对L-蛋氨酸具有非常高的降解活性、更好的血清稳定性和高选择性的胱硫氨酸-g-裂解酶。2.通过位点特异性的聚乙二醇化或与长的内在无序多肽序列(XTEN)融合，建立延长循环中存留时间的“人L-蛋氨酸酶”的优化形式，并测定其药代动力学和药效学性质。3.评价这些酶在人神经母细胞瘤小鼠异种移植模型中的疗效，该模型是利用诊断或复发的临床细胞系形成的。这些酶的用途将作为单一疗法和与长春新碱联合治疗进行研究。 公共卫生相关性：神经母细胞瘤是儿童最常见的颅外实体肿瘤，在患有晚期疾病的儿童中，治愈率高达惊人的30%。显然需要新的化疗药物，因为目前的药物在导致毒性、急性和严重的长期副作用的大剂量下只有轻微的效果。我们正在开发一种由人类酶改造而成的药物，这种药物可以通过攻击神经母细胞瘤细胞的一个关键代谢点来杀死肿瘤，并在与目前低剂量的无毒化疗药物结合使用时显示出极大的希望。