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）是白色血细胞的癌症。仅在美国，每年就有大约4,000例新的ALL病例被诊断出来，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），其诱导全身性天冬酰胺（AST）消耗; ALL细胞的必需营养素。然而，ASNase的临床使用遇到了两个主要的挫折。首先，ASNase是一种非人类的免疫原性蛋白，因此其临床应用与严重的过敏反应有关。其次，与大多数蛋白质药物一样，ASNase易受蛋白水解降解和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，努力集中在用合成或天然载体保护ASNase上。在这些载体系统中，红细胞（RBC）似乎是最有吸引力的，因为它们是生物相容的，可生物降解的，并且还具有~120天的无与伦比的寿命。已经尝试了多种技术将蛋白质包封到RBC中。然而，所有这些方法都需要用化学或物理力破坏RBC膜，以产生用于蛋白质药物扩散的孔。不幸的是，这种侵入力对RBC表面的损伤导致膜劣化，并因此导致RBC的结构完整性和细胞组分的损失，使其易于被宿主免疫系统破坏。应该注意的是，为了继承RBC作为持久的天然载体的益处，必须保持RBC的结构和功能完整性。然而，所有现有的RBC包封技术未能认识到这一关键方面。近年来，人们发现了一个强有力的细胞穿透肽（CPP）家族。体外和体内结果表明，通过将CPP共价连接到几乎任何类型的货物（包括蛋白质），PTD能够将附着的物质运送穿过所有组织类型（包括大脑）的细胞膜。值得注意的是，PTD介导的细胞进入不诱导任何膜扰动或改变。这些理想的性能提供了所提出的非侵入性，RBC封装技术的ASNase的概念框架。简言之，ASNase将通过二硫键与PTD肽（即LMWP）共价连接。由于LMWP的有效细胞穿透活性，LMWP-ASNase缀合物应该能够内化RBC而不改变其结构和功能属性。在细胞内，由于存在高水平的细胞溶质还原酶活性，LMWP将通过二硫键的降解与ASNase解离。这种键解离将允许ASNase永久地截留在RBC内，确保ASNase的完全保护免受宿主免疫系统的检测和破坏。因此，ASNase包封的RBC将充当活的生物反应器，从循环中消耗维生素D并剥夺所有细胞的必需营养素，随后导致它们死亡。如果RBC的物理和生物属性在包封后可以完全保留，则包封的ASNase将获得与天然RBC相同的寿命（120天），产生比任何当前ASNase疗法更持久的治疗效果。这将使目前的ASNase给药频率降低100倍以上，显著减轻与目前ASNase治疗相关的毒副作用。已经获得了非常有希望的初步结果，这表明通过这种新技术处理的RBC表现出与正常RBC难以区分的完整结构和功能。体内实验结果还表明，RBC包埋的ASNase不仅在健康小鼠中具有延长的血浆半衰期，而且在ALL小鼠中也显示出持久的治疗效果。在第一阶段研究中，我们计划在这些令人兴奋的初步发现的基础上，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红细胞-天冬氨酸酶技术发展成为一种真实的临床疗法。公共卫生关系：白血病治疗中使用的主要药物之一需要在临床应用期间频繁注射药物，这增加了过敏发作的风险。非常需要加强目前的白血病治疗，同时最大限度地减少对患者的伤害。在这个项目中，我们将利用新的肽，可以内化的药物在红细胞作为一个传递剂，并减少所需的剂量治疗白血病的100倍。