Human engineered enzymes for L-Arg depletion chemotherapy

用于 L-Arg 耗竭化疗的人类工程酶

基本信息

批准号：
7636106
负责人：
GEORGE Georgiou GEORGIOU
金额：
$ 31.24万
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：
2009
资助国家：
美国
起止时间：
2009-03-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7636106
关键词：
Acute Lymphocytic Leukemia Adverse effects Alteplase Amino Acid Substitution Amino Acids Anabolism Animal Model Antibodies Arginine Arginine deiminase Asparagine Bacterial Proteins Bacteriophages Binding Biochemical Bioinformatics Blood Vessels Carbamoyl Transferases Carcinoma Cell Death Cell Line Cessation of life Chimeric Proteins Citrulline Clinical Clinical Data Clinical Trials Combined Modality Therapy Development Disease Dose Drug Kinetics Effectiveness Engineering Enzyme Kinetics Enzymes Epitopes Essential Amino Acids Europe Evaluation Exhibits FDA approved Fc domain Generations Goals Growth Half-Life Human Human Development Human Engineering Human Genome Hydrolysis Immune Immune response Immune system Immunoglobulin G Immunoglobulins In Vitro Injection of therapeutic agent International Isoenzymes Italy Kinetics Laboratories Lead Life Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of liver Measures Mediating Metabolic Methionine Modification Mucopolysaccharidosis I Mus Mutagenesis Mutation Non-Essential Amino Acid Non-Hodgkin&apos s Lymphoma Normal Cell Normal tissue morphology Nude Mice Ornithine Ornithine Carbamoyltransferase Orphan Drugs Pathway interactions Patients Pegaspargase Pharmacodynamics Phase Physiological Plasma Polymers Primary carcinoma of the liver cells Property Protein Engineering Protein-arginine deiminase Proteins Reaction Recycling Renal Cell Carcinoma Research S-1 Antimetabolite agent Serum Specificity Structure Substrate Specificity Techniques Testing Therapeutic Therapeutic Agents Tissues Translating Treatment Efficacy Variant Work Xenograft procedure arginase argininosuccinate synthase asparaginase bioprocess cancer cell cancer therapy catalyst cell growth cell killing chemotherapy clinical practice cofactor combinatorial coping cytotoxic cytotoxicity design efficacy evaluation experience high throughput screening immunogenic immunogenicity improved in vivo killings loved ones melanoma mouse model mutant novel strategies particle polypeptide pre-clinical public health relevance research clinical testing therapeutic protein tissue culture treatment strategy tumor

项目摘要

DESCRIPTION (provided by applicant): 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 enzyme for chemotherapy of hepatic cancers and melanomas by systemic L-Arginine depletion. Enzymatic depletion of arginine using PEGylated bacterial arginine deiminase (ADI) has been found to be of significant clinical benefit in the treatment of hepatoceullar carcinomas (HCC), renal cell carcinomas and melanoms. However the therapeutic utility of bacterial ADI enzymes is severely compromised by its high immunogenicity. We propose to engineer human enzymes that exhibit optimal catalytic, physical and pharmacokinetic properties without eliciting adverse immune responses. Combinatorial structure guided saturation mutagenesis, together with high throughput screening for arginine deiminase will be employed to generate two candidate enzymes: (i) mutants of peptidyl arginine deiminase 4 that hydrolyze L-Arginine instead of peptidyl arginine with high activity and (ii) engineered human Arginase variants exhibiting >10-fold lower KM for L-Arginine in plasma. The cytotoxic effect of these enzymes on various human HCC cell lines will be evaluated. Novel approaches for the modification of the engineered enzymes to achieved long serum persistence are described and these will be evaluated in mice. Finally, optimal dosing to achieve sustained depletion will be determined and tumor reduction and survival following administration will be assessed in human HCC xenografts. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinomas (HCC) kill hundreds of thousands of people worldwide every year. These cancers are very aggressive, and very difficult to treat, making new treatments of the utmost importance. However, one new treatment approach has recently been found that promises to keep these killers at bay. Hepatocellular carcinomas have lost the ability to make the amino acid L-Arginine, one of the building blocks necessary for cell growth. Cancer cells cope with this by scavenging L-Arginine from their surroundings and continue with unchecked growth. Excitingly though, when these tumors are treated with a bacterial enzyme that breaks down L-Arginine, these cancers starve to death while normal tissue is unharmed. The downside is that the body's immune system violently reacts to foreign particles, often making the treatment as dangerous as the disease. Our goal is take human enzymes and "tweak" them slightly so that they will efficiently break down L-Arginine. Dangerous immune responses will be avoided because these enzymes will be recognized as normal human proteins. In this proposal we will continue the engineering of two human enzymes (Peptidylarginine Deiminase and Arginase) that have already led to significant improvements in therapeutic potential. Using standard protein engineering techniques we will make highly active, and stable enzymes that will enable cancer cells to be specifically eliminated, without the harmful side-effects from using bacterial proteins. We believe that the research outlined here can create safe, effective, therapeutic agents for patients with hepatocellular carcinomas, and give our afflicted loved ones a second chance at life.

描述（由申请人提供）：本提案的总体目标是采用现代蛋白质工程技术来开发新一代非免疫原性和药理学优化的酶，用于通过全身性 L-精氨酸消耗来进行肝癌和黑色素瘤的化疗。已发现使用聚乙二醇化细菌精氨酸脱亚胺酶 (ADI) 酶促消耗精氨酸在肝细胞癌 (HCC)、肾细胞癌和黑色素瘤的治疗中具有显着的临床益处。然而，细菌 ADI 酶的治疗效用因其高免疫原性而受到严重损害。我们建议设计出具有最佳催化、物理和药代动力学特性且不会引起不良免疫反应的人类酶。将采用组合结构引导的饱和诱变以及精氨酸脱亚胺酶的高通量筛选来产生两种候选酶：(i) 肽基精氨酸脱亚胺酶 4 的突变体，其水解 L-精氨酸而不是具有高活性的肽基精氨酸和 (ii) 工程化人类精氨酸酶变体对血浆中 L-精氨酸的 KM 降低 >10 倍。将评估这些酶对各种人类 HCC 细胞系的细胞毒性作用。描述了修饰工程酶以实现长期血清持久性的新方法，并将在小鼠中对这些方法进行评估。最后，将确定实现持续消除的最佳剂量，并在人类 HCC 异种移植物中评估给药后的肿瘤减少和存活率。公共卫生相关性：肝细胞癌 (HCC) 每年导致全世界数十万人死亡。这些癌症非常具有侵袭性，并且非常难以治疗，因此新的治疗方法至关重要。然而，最近发现了一种新的治疗方法，有望阻止这些杀手。肝细胞癌已经失去了制造氨基酸 L-精氨酸的能力，L-精氨酸是细胞生长必需的组成部分之一。癌细胞通过从周围环境中清除L-精氨酸来应对这一问题，并继续不受抑制的生长。但令人兴奋的是，当用分解 L-精氨酸的细菌酶治疗这些肿瘤时，这些癌症会饿死，而正常组织却毫发无伤。缺点是人体的免疫系统会对异物产生剧烈反应，常常使治疗与疾病一样危险。我们的目标是采用人类酶并稍微“调整”它们，以便它们能够有效地分解 L-精氨酸。将避免危险的免疫反应，因为这些酶将被识别为正常的人类蛋白质。在这项提案中，我们将继续对两种人类酶（肽基精氨酸脱亚胺酶和精氨酸酶）进行工程改造，这两种酶已经显着提高了治疗潜力。使用标准蛋白质工程技术，我们将制造高活性且稳定的酶，使癌细胞能够被特异性消除，而不会产生使用细菌蛋白质带来的有害副作用。我们相信，这里概述的研究可以为肝细胞癌患者创造安全、有效的治疗药物，并为我们患病的亲人提供第二次生命的机会。