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（全部）是白细胞癌。仅在美国，每年大约有4,000例新病例被诊断出，其中60％在儿童中发现。在所有治疗中使用的主要药物之一是L-天冬酰胺酶（ASNase），它诱导天冬酰胺（ASN）的全身耗竭；所有细胞的必需营养素。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，因此其临床用途与主要的过敏反应有关。其次，与大多数蛋白质药物一样，ASNase易受蛋白水解降解并清除。结果，ASNase的血浆半衰期相当短（〜25hr），因此要求频繁注射该药物，以进一步增加过敏性攻击的风险。为了克服此类问题，努力一直集中在使用合成或天然载体的ASNase上。在这些载体系统中，红细胞（RBC）似乎最吸引人，因为它们具有生物相容性，可生物降解，并且具有〜120天的无与伦比的寿命。已经尝试将蛋白质封装到RBC中。 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.应当指出的是，为了继承RBC作为持久的自然载体的好处，必须保留RBC的结构和功能完整性。但是，所有现有的RBC封装技术都无法识别这一关键方面。最近，已经发现了一个有效的细胞穿透肽（CPP）家族。在体外和体内结果表明，通过将CPP与几乎所有类型的Cargos联系起来，PTD能够将CPP连接到包括大脑在内的所有组织类型（包括大脑）的细胞膜之间的附着物种。值得注意的是，PTD介导的细胞进入不会诱导任何膜扰动或改变。这些理想的属性为ASNase提出的非侵入性，RBC封装技术提供了概念框架。简而言之，ASNase将通过二硫键连接与PTD肽（即LMWP）共价链接。由于LMWP的有效细胞渗透活性，LMWP酶共轭物应能够在不改变其结构和功能属性的情况下内化RBC。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分离。这种债券解离将使ASNase永久捕获RBC，以确保对ASNase的全面保护免受宿主免疫系统的检测和破坏。因此，ASNase已封装的RBC将起到活物生物反应器的作用，从循环中耗尽ASN，并剥夺了所有细胞的必需营养素，随后导致其死亡。如果封装后可以完全保留RBC的物理和生物学特性，则包含的ASNase然后将其加入到天然RBC的相同寿命（120天）（120天），从而产生比任何当前ASNase疗法的持久治疗效应。这将使当前的Asnase剂量频率降低100倍以上，从而显着减轻与当前的ASNase疗法相关的毒性副作用。已经获得了极其有希望的初步结果，该结果表明，这项新技术处理的RBC表现出完整的结构和功能，与正常RBC无法区分。体内的结果还表明，加拿大rbc纳入的ASNase不仅在健康小鼠中遗传了长时间的血浆半衰期，而且在全捕获小鼠中表现出持久的治疗作用。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，并进行概念验证的动物调查，以进一步验证这项技术。我们的最终目标是将这种RBC ASNase技术发展为真正的临床补救措施。公共卫生疗效：白血病治疗中使用的主要药物之一需要在临床应用过程中要求和频繁注射该药物，以增加过敏攻击的风险。非常需要增强当前的白血病疗法，同时最大程度地减少对患者的伤害。在这个项目中，我们将利用可以将红细胞中药物内化为递送剂的新型肽，并减少将白血病治疗所需的剂量减少100倍。