RBC_Encapsulated Asparaginase for Enhanced Acute Lymphoblastic Leukemia Therapy

RBC_封装天冬酰胺酶用于增强急性淋巴细胞白血病治疗

• 批准号: 7538982
• 负责人: Allan E. David
• 金额: $ 12.41万
• 依托单位: INDUSTRIAL SCIENCE & TECHNOLOGY NETWORK
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-12 至 2010-03-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7538982
• 关键词: Acute; Acute Lymphocytic Leukemia; Adolescent; Adverse effects; Agreement; Allergic; Amino Acids; Animals; Asparagine; Biocompatible; Biological; Bioreactors; Blood Circulation; Brain; Cell Death; Cell Membrane Alteration; Cell membrane; Cell physiology; Cell surface; Cells; Cessation of life; Chemicals; Child; Clinical; Detection; Deterioration; Diagnosis; Dialysis procedure; Diffuse; Disruption; Dissociation; Disulfide Linkage; Dose; Drug Carriers; Drug Kinetics; Drug usage; Electroporation; Encapsulated; Endocytosis; Endogenous Factors; Endopeptidases; Ensure; Erythrocytes; Evaluation; Exhibits; Face; Family; Frequencies; Glutathione Reductase; Goals; Guanosine Monophosphate; Half-Life; Histocompatibility Testing; Hour; Human; Immune system; In Vitro; Infection; Inherited; Injection of therapeutic agent; Invasive; Investigation; Leukemic Cell; Leukocytes; Life; Link; Longevity; Lymphoblastic Leukemia; Malignant Neoplasms; Mechanics; Mediating; Membrane; Methods; Michigan; Molecular Weight; Mus; Nutrient; Object Attachment; Organ; Osmosis; Oxidoreductase; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Pharmacodynamics; Phase; Physical Dialysis; Plasma; Polymers; Pore Proteins; Preparation; Procedures; Process; Property; Protamines; Proteins; Public Health; Range; Recombinants; Reducing Agents; Research; Research Project Grants; Reticuloendothelial System; Risk; Safety; Scientist; Serum; Structure; Surface; System; Techniques; Technology; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Effect; Toxic effect; United States; Universities; Yang; age group; asparaginase; clinical application; concept; day; design; disulfide bond; fighting; immunogenic; immunogenicity; in vivo; leukemia; new technology; novel; response

DESCRIPTION (provided by applicant): Acute lymphoblastic leukemia (ALL) is cancer of white blood cells. Approximately 4,000 new cases of ALL are diagnosed annually in the US alone, with 60% of them found in children. One of the major drugs used in ALL treatment is L-asparaginase (ASNase), which induces a systemic depletion of asparagine (ASN); an essential nutrients for ALL cells. Nevertheless, clinical use of ASNase encounters two major setbacks. First, ASNase is a non-human, immunogenic protein, and its clinical use is thus associated with major anaphylactic responses. Secondly, like most protein drugs, ASNase is susceptible to proteolytic degradation and RES clearance. As a result, plasma half-life of ASNase is rather short (~25hr), thereby demanding frequent injections of the drug that further increase the risk of allergic attack. To overcome such problems, efforts have been focused on protection of ASNase with a synthetic or natural carrier. Among these carrier systems, red blood cells (RBC) appear to be most appealing, because they are biocompatible, biodegradable, and also possess an unmatched life-span of ~120 days. A variety of techniques has been attempted to encapsulate proteins into RBC. However, all of these methods require disruption of RBC membrane with a chemical or physical force to create pores for proteins drugs to diffuse in. Unfortunately, insult on the RBC surface by such an invasive force causes membrane deterioration and, consequently, results in a loss of structural integrity and cellular components of the RBC, rendering it prone to destruction by the host immune system. It should be noted that in order to inherit the benefits of RBC as a long-lasting, natural carrier, it is essential to retain both structural and functional integrity of RBC. Yet, all of the existing RBC encapsulation techniques fail to recognize this critical aspect. Recently, a family of potent cell-penetrating peptides (CPP) has been discovered. In vitro and in vivo results revealed that, by covalently linking CPP to almost any type of cargos including proteins, PTD was able to ferry the attached species across cell membranes of all tissue types, including the brain. Remarkably, PTD-mediated cell entry does not induce any membrane perturbation or alteration. These desirable properties provide the conceptual framework of the proposed non-invasive, RBC-encapsulation technology for ASNase. Briefly, ASNase will be covalently linked with a PTD peptide (i.e. LMWP) via a disulfide linkage. Due to the potent cell-penetrating activity of LMWP, the LMWP-ASNase conjugates should be able to internalize a RBC without altering its structural and functional attributes. Within the cell, LMWP would be dissociated from ASNase via degradation of the disulfide bond, due to the presence of a high level of cytosolic reductase activity. This bond dissociation would allow ASNase to remain permanently entrapped within RBC, ensuring a full protection of ASNase from detection and destruction by the host immune system. Hence, the ASNase-encapsulated RBC would function as a live bioreactor, depleting ASN from the circulation and depriving ALL cells of essential nutrients, subsequently leading to their deaths. If both of the physical and biological attributes of RBC can be fully retained after encapsulation, the entrapped ASNase would then accede to the same life-span of native RBC (120 days), yielding the longest lasting therapeutic effects than any current ASNase therapies. This would reduce current ASNase dosing frequency by more than 100 folds, significantly alleviating the toxic side effects associated with present ASNase therapies. Extremely promising preliminary results have been obtained, which showed RBC processed by this novel technology exhibited an intact structure and functionality that were indistinguishable from normal RBC. In vivo results also showed that RBC-entrapped ASNase not only inherited a prolonged plasma half-life in healthy mice but also displayed a long-lasting therapeutic effects in ALL-harboring mice. In this Phase I research, we plan to build on these exciting preliminary findings and carry out a proof-of-concept animal investigation to further validate this technology. Our ultimate goal is to develop this RBC- ASNase technology into a real clinical remedy. PUBLIC HEALTH RELAVENCE:One of the major drugs used in leukemia treatment requires demanding and frequent injections of the drug during clinical application that increases the risk of allergic attack. There is a great need to enhance current leukemia therapy while minimizing harm to the patient. In this project we will utilize novel peptides that can internalize the drug in red blood cells as a delivery agent and reduce the dose required to treat leukemia by 100 fold.

描述（由申请人提供）： 急性淋巴细胞白血病（ALL）是白细胞癌症。仅在美国每年就诊断出约 4,000 例新的 ALL 病例，其中 60% 是儿童。 ALL 治疗中使用的主要药物之一是 L-天冬酰胺酶 (ASNase)，它会引起天冬酰胺 (ASN) 的全身性消耗；所有细胞的必需营养素。然而，ASNase 的临床使用遇到了两个重大挫折。首先，ASNase 是一种非人类免疫原性蛋白质，因此其临床应用与主要的过敏反应相关。其次，与大多数蛋白质药物一样，ASNase 容易受到蛋白水解降解和 RES 清除的影响。因此，ASNase 的血浆半衰期相当短（约 25 小时），因此需要频繁注射药物，这进一步增加了过敏发作的风险。为了克服这些问题，人们一直致力于用合成或天然载体保护 ASNase。在这些载体系统中，红细胞 (RBC) 似乎最具吸引力，因为它们具有生物相容性、可生物降解性，并且还具有无与伦比的约 120 天的寿命。人们尝试了多种技术将蛋白质封装到红细胞中。然而，所有这些方法都需要用化学或物理力破坏红细胞膜，以形成供蛋白质药物扩散的孔。不幸的是，这种侵入力对红细胞表面的损伤会导致膜退化，从而导致损失红细胞的结构完整性和细胞成分，使其容易被宿主免疫系统破坏。应该指出的是，为了继承红细胞作为持久、天然载体的优势，必须保留红细胞的结构和功能完整性。然而，所有现有的红细胞封装技术都未能认识到这一关键方面。最近，发现了一个有效的细胞穿透肽（CPP）家族。体外和体内结果表明，通过将 CPP 与包括蛋白质在内的几乎任何类型的货物共价连接，PTD 能够将附着的物质运送到包括大脑在内的所有组织类型的细胞膜上。值得注意的是，PTD 介导的细胞进入不会引起任何膜扰动或改变。这些理想的特性为所提出的 ASNase 非侵入性红细胞封装技术提供了概念框架。简而言之，ASNase 将通过二硫键与 PTD 肽（即 LMWP）共价连接。由于 LMWP 具有强大的细胞穿透活性，LMWP-ASNase 缀合物应该能够内化红细胞而不改变其结构和功能属性。在细胞内，由于存在高水平的胞质还原酶活性，LMWP 将通过二硫键的降解与 ASNase 解离。这种键解离将使 ASNase 能够永久地被捕获在红细胞内，确保充分保护 ASNase 免受宿主免疫系统的检测和破坏。因此，ASNase 封装的红细胞将起到活生物反应器的作用，从循环中耗尽 ASN，并剥夺所有细胞的必需营养，随后导致细胞死亡。如果封装后红细胞的物理和生物学属性都能得到充分保留，那么被包埋的 ASNase 将获得与天然红细胞相同的寿命（120 天），从而产生比任何现有 ASNase 疗法更持久的治疗效果。这将使目前的 ASNase 给药频率减少 100 倍以上，从而显着减轻与现有 ASNase 疗法相关的毒副作用。已经获得了非常有希望的初步结果，表明通过这种新技术处理的红细胞表现出完整的结构和功能，与正常红细胞没有区别。体内结果还表明，红细胞包埋的 ASNase 不仅在健康小鼠中继承了较长的血浆半衰期，而且在携带 ALL 的小鼠中也显示出持久的治疗效果。在这一阶段的研究中，我们计划以这些令人兴奋的初步发现为基础，进行概念验证动物研究，以进一步验证这项技术。我们的最终目标是将这种 RBC-ASNase 技术开发成真正的临床疗法。公共卫生相关性：用于白血病治疗的主要药物之一在临床应用过程中需要频繁且严格的注射，这增加了过敏发作的风险。非常需要加强当前的白血病治疗，同时尽量减少对患者的伤害。在这个项目中，我们将利用新型肽作为递送剂，将药物内化到红细胞中，并将治疗白血病所需的剂量减少 100 倍。