Human engineered enzymes for L-Arg depletion chemotherapy

描述(申请人提供)：本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，用于全身L-精氨酸耗竭治疗肝癌和黑色素瘤。聚乙二醇化细菌精氨酸脱亚氨酶(ADI)酶促精氨酸脱除在治疗肝细胞癌、肾细胞癌和黑色素瘤方面有显著的临床疗效。然而，细菌ADI酶的高免疫原性严重影响了其治疗作用。我们建议设计人类酶，使其具有最佳的催化、物理和药代动力学特性，而不会引起不良免疫反应。组合结构引导的饱和突变和精氨酸脱亚胺酶的高通量筛选将被用来产生两种候选酶：(I)能高活性地水解L-精氨酸而不是肽基精氨酸的肽基精氨酸脱亚胺酶4突变体；(Ii)对L-精氨酸的Km降低10倍的人精氨酸酶工程化突变体。将评估这些酶对各种人肝癌细胞系的细胞毒作用。描述了对工程酶进行修饰以实现长时间血清持久性的新方法，这些方法将在小鼠身上进行评估。最后，将确定实现持续耗竭的最佳剂量，并将评估给药后人肝癌移植瘤的肿瘤减少和存活率。与公共卫生相关：全球每年有数十万人死于肝细胞癌。这些癌症侵袭性很强，很难治疗，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，这是细胞生长所必需的组成部分之一。癌细胞通过清除周围环境中的L-精氨酸来应对这种情况，并继续不受限制地生长。然而，令人兴奋的是，当用一种分解L-精氨酸的细菌酶治疗这些肿瘤时，这些癌症会饿死，而正常组织却没有受到伤害。缺点是，人体的免疫系统会对外来颗粒做出激烈的反应，往往会使治疗变得与疾病一样危险。我们的目标是利用人类的酶，对它们进行微调，这样它们就能有效地分解L-精氨酸。危险的免疫反应将被避免，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)进行工程设计，这两种酶已经在治疗潜力方面取得了显着的改进。使用标准的蛋白质工程技术，我们将制造高度活性和稳定的酶，使癌细胞能够被特定地消除，而不会产生使用细菌蛋白质的有害副作用。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，并给我们痛苦的亲人第二次生命。

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.