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.

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 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.此外，还报道了与微管解聚剂的强烈协同效应。 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 (c) 在小鼠体内的稳定性提高了约 10 倍。 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.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。