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利用现代蛋白质工程技术开发新一代非免疫原性和药理学优化的酶，用于通过全身l -精氨酸消耗来化疗肝癌和黑色素瘤。使用聚乙二醇化细菌精氨酸脱亚胺酶（ADI）去除精氨酸已被发现在治疗肝细胞癌（HCC）、肾细胞癌和黑色素瘤中具有显著的临床益处。然而，细菌ADI酶的治疗效用受到其高免疫原性的严重损害。我们建议设计人类酶，表现出最佳的催化，物理和药代动力学性质，而不会引起不良的免疫反应。组合结构引导饱和诱变，结合精氨酸脱亚胺酶的高通量筛选，将产生两种候选酶：(i)水解l -精氨酸的肽基精氨酸脱亚胺酶4突变体，而不是高活性的肽基精氨酸；（ii）工程的人类精氨酸酶变异体，其在血浆中对l -精氨酸的KM降低了10倍。这些酶对各种人类HCC细胞系的细胞毒性作用将被评估。描述了修饰工程酶以实现长血清持久性的新方法，这些方法将在小鼠中进行评估。最后，将确定实现持续耗竭的最佳剂量，并评估人类肝癌异种移植物给药后的肿瘤减少和生存。公共卫生相关性：肝细胞癌（HCC）每年导致全球数十万人死亡。这些癌症具有很强的侵袭性，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l -精氨酸的能力，而l -精氨酸是细胞生长所必需的基本成分之一。癌细胞通过从周围环境中清除l -精氨酸来应对这种情况，并继续无限制地生长。然而令人兴奋的是，当这些肿瘤被一种分解l -精氨酸的细菌酶治疗时，这些癌细胞饿死了，而正常组织却毫发无损。缺点是身体的免疫系统会对外来颗粒产生强烈的反应，通常会使治疗和疾病本身一样危险。我们的目标是把人类的酶稍微“调整”一下，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。在这个提议中，我们将继续工程的两种人类酶（肽精氨酸脱亚胺酶和精氨酸酶），已经导致显著改善治疗潜力。使用标准的蛋白质工程技术，我们将制造出高活性、稳定的酶，使癌细胞能够被特异性地消灭，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，并给我们受折磨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.