Targeting toxins to tumors using microproteins

使用微生物蛋白将毒素靶向肿瘤

• 批准号: 7687366
• 负责人: Volker Schellenberger
• 金额: $ 46.33万
• 依托单位: AMUNIX OPERATING, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-06 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7687366
• 关键词: Address; Adverse effects; Affinity; Amino Acid Sequence; Antibodies; Antibody Specificity; Binding; Blood Platelets; Cells; Characteristics; Chemicals; Chimeric Proteins; Clinical; Complex; Denileukin Diftitox; Development; Disintegrins; Dose; Drug Kinetics; Elements; Engineering; Escherichia coli; Evaluation; Genes; Goals; Health; In Vitro; Integrins; Lead; Length; Manufacturer Name; Minor; Modification; Neoplasm Metastasis; Patients; Peptide Sequence Determination; Performance; Phage Display; Pharmaceutical Preparations; Pharmacodynamics; Phase; Polyethylene Glycols; Production; Property; Proteins; Protocols documentation; Route; Serum; Site; Small Business Innovation Research Grant; Specificity; Targeted Toxins; Testing; Therapeutic; Therapeutic antibodies; Toxic effect; Treatment Protocols; Tumor Tissue; base; cell killing; chemical property; chemotherapy; cytotoxic; immunogenic; immunogenicity; in vivo; killings; manufacturing process; meetings; neoplastic cell; novel; pre-clinical; protein aggregation; public health relevance; success; tumor; tumor specificity

DESCRIPTION (provided by applicant): Treatment of metastatic tumors is a major health challenge. Recently, antibody-based therapies have been developed that are more specific and have fewer side effects compared with conventional chemotherapy. However, the potency of most antibody therapeutics is limited by their inadequate ability to kill tumor cells. Consequently, there is an urgent, unmet need to develop therapeutics that combine the specificity of antibodies for tumor tissues with a potent cytotoxic function. The development of tumor-targeted toxins has yielded promising results and led to one approved product, Ontak (Denileukin). Most molecules however are immunogenic and aggregation prone, have limited stability and require complex manufacturing routes. To achieve clinical and commercial success it is critical for candidates to meet following criteria: 1) high potency; 2) low systemic toxicity; 3) low immunogenicity; 4) high protein stability, lack of aggregation; 5) robust manufacturing. This proposal aims to develop tumor-targeted toxins by combining three elements that confer significant advantages over current approaches: microproteins for tumor binding/internalization; RNAse for cell killing; rPEG to optimize PK properties. In the successful Phase I of this project, we developed tumor-specific microproteins with the following properties: 1) efficient production in E. coli; 2) efficient phage display that enables rapid specificity optimization; 3) effective internalization of toxic payloads; 4) excellent serum stability. In a separate phase I SBIR project, we developed rPEGs, hydrophilic protein sequences that mimic the properties of chemical polyethylene glycol (PEG) but can be directly fused to other proteins. rPEGs optimize the pharmacokinetics of a product, reduce product immunogenicity, and greatly reduce protein aggregation. Our Phase II goal is to optimize the specificity of our lead microproteins to achieve a >1000x ration of tumor/normal affinity. Subsequently, we will fuse these optimized microproteins to RNAse as toxic payload and rPEG to optimize PK, PD and protein manufacturing. The resulting fusion proteins will be thoroughly evaluated for in vitro and in vivo performance. In addition, we will develop an effective manufacturing process that can be transferred with minor modifications to a GMP manufacturer. We aim to generate two lead molecules that will be ready to enter preclinical followed by clinical development. In addition we will generate microprotein-rPEG fusions with defined conjugation sites that will be uniquely suitable for the chemical conjugation of toxic payloads. PUBLIC HEALTH RELEVANCE: The development of tumor-targeted toxins have yielded promising results and led to one approved product, Denileukin. However, existing molecules have significant limitations especially immunogenicity and complex manufacturing requirements. This project will use tumor-specific microproteins to address these limitations and develop targeted toxins with the following characteristics: 1) high potency; 2) low systemic toxicity; 3) low immunogenicity to allow repeat dosing; 4) good protein stability; 5) lack of aggregation; 6) robust manufacturing process.

描述(申请人提供)：转移性肿瘤的治疗是一个主要的健康挑战。最近，基于抗体的治疗方法已经被开发出来，与传统的化疗相比，这种治疗方法更具特异性，副作用更少。然而，大多数抗体疗法的效力受到它们杀死肿瘤细胞能力不足的限制。因此，迫切需要开发一种将肿瘤组织抗体的特异性与强大的细胞毒功能相结合的疗法。针对肿瘤的毒素的开发已经产生了有希望的结果，并导致了一种批准的产品，Ontak(Denileukin)。然而，大多数分子是免疫原性的，容易聚集，稳定性有限，需要复杂的制造工艺。为了获得临床和商业上的成功，候选药物必须满足以下标准：1)高效力；2)低系统毒性；3)低免疫原性；4)高蛋白质稳定性，缺乏聚集性；5)稳健的制造。这项提议旨在通过结合三种元素来开发肿瘤靶向毒素，这三种元素与目前的方法相比具有显著的优势：用于肿瘤结合/内化的微蛋白；用于细胞杀伤的核糖核酸酶；用于优化PK特性的rPEG。在该项目成功的第一阶段，我们开发了具有以下特性的肿瘤特异性微蛋白：1)在大肠杆菌中高效生产；2)高效噬菌体展示，使特异性快速优化；3)有效内化有毒有效载荷；4)良好的血清稳定性。在另一个单独的第一阶段SBIR项目中，我们开发了RPEGS，这是一种亲水性蛋白质序列，模仿化学聚乙二醇(PEG)的性质，但可以直接与其他蛋白质融合。RPEGs优化了产品的药代动力学，降低了产品的免疫原性，并大大减少了蛋白质的聚集。我们的第二阶段目标是优化我们的铅微蛋白的特异性，以实现肿瘤/正常亲和力的1000倍比率。随后，我们将这些优化的微蛋白融合到RNase作为有毒有效载荷和rPEG来优化PK、PD和蛋白质制造。由此产生的融合蛋白将在体外和体内进行彻底的性能评估。此外，我们将开发一种有效的制造工艺，只需稍加修改即可转移给GMP制造商。我们的目标是产生两个领先分子，它们将准备进入临床前，随后进行临床开发。此外，我们将产生具有特定结合位点的微蛋白-rPEG融合，这将是唯一适合有毒有效载荷的化学结合的融合。 与公共卫生相关：肿瘤靶向毒素的开发取得了令人振奋的结果，并导致了一种批准的产品--Denileukin。然而，现有的分子有很大的局限性，特别是免疫原性和复杂的制造要求。该项目将使用肿瘤特异性微蛋白来解决这些局限性，并开发具有以下特征的靶向毒素：1)高效力；2)低系统毒性；3)低免疫原性，允许重复给药；4)良好的蛋白质稳定性；5)缺乏聚集；6)稳健的制造工艺。