Human engineered enzymes for L-Arg depletion chemotherapy

描述（由申请人提供）：本提案的总体目标是采用蛋白质工程的现代技术开发新一代非免疫原性和酶优化的酶，用于通过全身性L-精氨酸消耗进行肝癌和黑素瘤的化疗。已发现使用PEG化细菌精氨酸脱亚胺酶（ADI）的精氨酸酶促消耗在肝细胞癌（HCC）、肾细胞癌和黑色素瘤的治疗中具有显著的临床益处。然而，细菌ADI酶的治疗效用由于其高免疫原性而严重受损。我们建议工程人类酶，表现出最佳的催化，物理和药代动力学特性，而不会引发不良的免疫反应。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）工程化的人精氨酸酶变体，其对血浆中的L-精氨酸表现出>10倍的低KM。将评价这些酶对各种人HCC细胞系的细胞毒性作用。新的方法的工程酶的修饰，以实现长血清持久性进行了描述，这些将在小鼠中进行评估。最后，将确定实现持续消耗的最佳剂量，并将在人HCC异种移植物中评估施用后的肿瘤减少和存活。公共卫生相关性：全世界每年有数十万人死于肝细胞癌（HCC）。这些癌症非常具有侵略性，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-精氨酸是细胞生长所必需的组成部分之一。癌细胞通过从周围环境中清除L-精氨酸来科普这一点，并继续不受控制的生长。令人兴奋的是，当这些肿瘤用一种分解L-精氨酸的细菌酶治疗时，这些癌症会饿死，而正常组织则不会受到伤害。缺点是身体的免疫系统对外来颗粒产生强烈反应，通常使治疗与疾病一样危险。我们的目标是采取人类酶和“微调”他们稍微，使他们将有效地分解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。在本提案中，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，这两种酶已经导致了治疗潜力的显著改善。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，使癌细胞能够被特异性地消除，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，并给我们受苦的亲人第二次生命的机会。

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.