ENGINEERING THERAPEUTIC PROTEASES

工程治疗性蛋白酶

• 批准号: 8201010
• 负责人: ROBERT F BALINT
• 金额: $ 29.98万
• 依托单位: CYTODESIGN, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201010
• 关键词: Acceleration; Amino Acid Sequence; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antibody Affinity; Antigens; Attenuated; Binding Sites; Biological Assay; Biosensor; Cardiovascular system; Cell Culture Techniques; Cells; Chymase; Cleaved cell; Clinic; Communicable Diseases; Computing Methodologies; Custom; Disease; Dose; Effectiveness; Engineering; Enzymes; Escherichia coli; Family; Galium; Goals; Human; Immunoglobulin Idiotypes; In Vitro; Inflammation; Inflammatory; Kinetics; Left; Length; Libraries; Ligands; Malignant Neoplasms; Modeling; Monoclonal Antibodies; Mutagenesis; Mutation; Pathology; Peptide Hydrolases; Peptide Sequence Determination; Phase; Phenotype; Process; Property; Protease Domain; Protein Engineering; Reaction; Recovery; Safety; Screening procedure; Serine Protease; Substrate Specificity; Surface; System; Systems Development; TNF gene; Testing; Therapeutic; Validation; Variant; base; chymotrypsin; design; design and construction; enzyme structure; family structure; human disease; immunogenicity; improved; knowledge base; programs; receptor; scaffold; sensor

DESCRIPTION (provided by applicant): Disregulated inflammation underlies or exacerbates the pathology of many human diseases, and an effective anti-inflammatory strategy involves stoichiometric antagonism of one or more components of the large network of pro-inflammatory receptor-ligand interactions. Monoclonal antibodies (mAb) have proven effective in the clinic against inflammatory targets. However, stoichiometric activity has often limited their effectiveness by necessitating the use of large doses to achieve adequate efficacy. Also, the potential immunogenicity of unnaturally high titers of single anti-self idiotypes often leads to a loss of efficacy. Such limitations could be overcome if the catalytic power of enzymes could be harnessed for the treatment of inflammatory disease. Custom design of enzymatic activities for desired applications has long been a major goal of protein engineering. However, current computational methods for the rational alteration of enzyme structures to enable desired reactions still leave prohibitively large amounts of protein sequence space to be searched for the required mutations. As a result, progress has been hampered by a lack of robust systems capable of efficient identification and recovery of rare variants possessing the sought activity from large libraries. To facilitate the engineering of target-specific proteolytic enzymes, a proprietary proteolytic activity sensor has been developed, which when co-expressed in cells with mutagenic libraries of a proteolytic scaffold, confers a selectable phenotype on cells expressing target-cleaving variants. The chymotrypsin family of serine proteases includes many of the most intensively studied of all enzymes. Thus, the chymotrypsin family structure/activity knowledge base permits the mutagenic targeting of residues lining the substrate binding sites to create a broad range of substrate specificities that can be efficiently searched with the aforementioned proteolytic activity biosensor. The goal of the proposed program is two-fold: (1) validate and optimize the proteolytic activity biosensor and protease library design and construction for selection and activity maturation of a human chymotrypsin-like protease against a human inflammatory target with a preliminary kcat/Km value of at least 102 M-1sec-1, and (2) test the protease for bioactivity in vitro. Human TNF1 will be used as a model target for development of the system because it is clinically proven for the treatment of inflammatory diseases, against which TNF1-neutralizing proteases can be tested and compared with approved anti-TNF1 antibodies and other stoichiometric therapeutics. The sensor will incorporate full-length human TNF1 for selections from a mutagenic library based on the proteolytic domain of HtrA1, a human chymotrypsin family protease with unique properties for the engineering of target-specific activities. The proposed program is expected to take 16 months, at the end of which we hope to have established (1) optimum selection conditions, (2) some indication of the range of proteolytic activities achievable with this system, and (3) how optimized target-cleaving proteases compare with high-affinity antibodies against the same target with respect to target neutralization in vitro. PUBLIC HEALTH RELEVANCE: The proposed project aims to develop a new system to solve a long-standing problem in protein engineering, namely to harness the catalytic power of enzymes for human therapy by engineering target-specific proteolytic activities into existing human protease scaffolds, which promises to provide improved efficacy and safety for the treatment of a broad range of diseases such as cancer, cardiovascular, inflammatory, and infectious diseases.

描述（由申请人提供）：失调的炎症是许多人类疾病病理的基础或加剧，有效的抗炎策略涉及促炎受体-配体相互作用的大网络的一种或多种组分的化学计量拮抗作用。单克隆抗体（mAb）已被证明在临床上有效对抗炎症靶点。然而，化学计量活性通常限制了它们的有效性，因为必须使用大剂量才能达到足够的功效。此外，单一抗自身独特型的非天然高滴度的潜在免疫原性通常导致疗效丧失。如果能利用酶的催化能力来治疗炎症性疾病，就可以克服这些局限性。为所需应用定制酶活性设计长期以来一直是蛋白质工程的主要目标。然而，目前用于合理改变酶结构以实现所需反应的计算方法仍然留下大量的蛋白质序列空间以搜索所需的突变。因此，由于缺乏能够从大型文库中有效鉴定和回收具有所需活性的稀有变体的稳健系统，进展受到阻碍。为了促进靶特异性蛋白水解酶的工程化，已经开发了专有的蛋白水解活性传感器，其当在细胞中与蛋白水解支架的诱变文库共表达时，赋予表达靶切割变体的细胞可选择的表型。丝氨酸蛋白酶的胰凝乳蛋白酶家族包括所有酶中研究最深入的许多酶。因此，胰凝乳蛋白酶家族结构/活性知识库允许对衬在底物结合位点上的残基进行诱变靶向，以产生可以用前述蛋白水解活性生物传感器有效搜索的宽范围的底物特异性。拟议计划的目标有两个：（1）验证和优化蛋白水解活性生物传感器和蛋白酶库的设计和构建，以选择和活性成熟针对人类炎症靶点的人类胰凝乳蛋白酶样蛋白酶，初步kcat/Km值至少为102 M-1秒-1，以及（2）测试蛋白酶的体外生物活性。人TNF 1将被用作开发该系统的模型靶标，因为它在临床上被证明用于治疗炎性疾病，可以测试TNF 1中和蛋白酶并与批准的抗TNF 1抗体和其他化学计量治疗剂进行比较。该传感器将包含全长人TNF 1，用于从基于HtrA 1蛋白水解结构域的致突变库中进行选择，HtrA 1是一种人胰凝乳蛋白酶家族蛋白酶，具有独特的靶向特异性活性工程特性。该计划预计需要16个月，在此结束时，我们希望已经建立（1）最佳选择条件，（2）用该系统可实现的蛋白水解活性范围的一些指示，以及（3）优化的靶切割蛋白酶如何与针对相同靶标的高亲和力抗体在体外靶中和方面进行比较。 公共卫生相关性：该项目旨在开发一种新的系统来解决蛋白质工程中的一个长期存在的问题，即通过将目标特异性蛋白水解活性工程化到现有的人类蛋白酶支架中，利用酶的催化能力进行人类治疗，这有望为治疗癌症，心血管，炎症和传染病等广泛疾病提供更好的疗效和安全性。