Targeting toxins to tumors using microproteins

描述（由申请人提供）：转移性肿瘤的治疗是一个主要的健康挑战。最近，以抗体为基础的治疗方法已经开发出来，与传统的化疗相比，它更具特异性，副作用更少。然而，大多数抗体疗法的效力受到其杀死肿瘤细胞的能力不足的限制。因此，迫切需要开发一种治疗方法，将抗体对肿瘤组织的特异性与有效的细胞毒性功能结合起来。肿瘤靶向毒素的开发已经取得了可喜的成果，并导致了一种获批产品Ontak （Denileukin）。然而，大多数分子具有免疫原性和聚集性，稳定性有限，需要复杂的制造路线。为了取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；2)低全身毒性；3)免疫原性低；4)蛋白质稳定性高，缺乏聚集性；5)稳健的制造业。该提案旨在通过结合三种元素来开发肿瘤靶向毒素，这些元素赋予了当前方法的显着优势：用于肿瘤结合/内化的微蛋白；杀死细胞的RNAse；rPEG优化PK性能。在该项目成功的第一期中，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，可快速优化特异性；3)有毒有效载荷的有效内化；4)优良的血清稳定性。在一个独立的一期SBIR项目中，我们开发了rpeg，这是一种亲水性蛋白质序列，它模仿化学聚乙二醇（PEG）的性质，但可以直接与其他蛋白质融合。rpeg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聚集。我们的II期目标是优化我们的先导微蛋白的特异性，以达到肿瘤/正常亲和力的1000倍。随后，我们将这些优化的微蛋白作为毒性载荷与RNAse和rPEG融合，以优化PK， PD和蛋白质制造。所得到的融合蛋白将被全面评估其在体外和体内的性能。此外，我们将开发一种有效的生产工艺，可以通过微小的修改转移到GMP制造商。我们的目标是产生两种先导分子，准备进入临床前，然后进行临床开发。此外，我们将生成具有确定的偶联位点的微蛋白- rpeg融合物，这将独特地适用于有毒有效载荷的化学偶联。

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.