Discovery of therapeutic antibodies targeting breast cancer metastasis using Enabled G-Protein-Coupled Receptors

项目摘要/摘要 Abilita Bio(AB)，一家创新驱动的生物技术公司，申请SBIR第一阶段资金 针对G蛋白偶联受体的新型治疗性抗体的发现 转移性乳腺癌。 在癌症治疗中，每种治疗方式都有自己的局限性。临床研究明确表明， 使用检查点抑制剂(PD-1/PD-L1、CTLA-4等)进行免疫治疗可以实现持久的免疫 改善临床结果的反应。然而，这种方法在给药时尤其有效。 与靶向治疗相结合，针对肿瘤扩散、归巢的基本组成部分 和生长，特别是在转移性癌症中。转移是限制癌症患者生存的主要因素。 因此，干扰控制肿瘤细胞转移行为的分子机制是 对于成功治疗癌症来说是必不可少的。 实验和临床证据支持这一概念，即最重要的机制之一 在转移过程中的操作涉及到稳态趋化因子及其受体。乳腺癌是 特点是具有明显的区域淋巴结、骨髓、肺和 肝脏。肿瘤细胞从原发肿瘤部位向转移归巢部位的迁移有许多相似之处 与白细胞贩运有关，这是由趋化因子结合的GPCRs严格控制的。有趣的是，有几个 研究证实，乳腺癌细胞分泌前列腺素E_2(PGE_2)可上调 通过激活前列腺素结合的GPCRs、EP2和EP4来表达CCR7。这项提案的重点是 靶向与肿瘤细胞转移和器官特异性侵袭密切相关的关键GPCRs 治疗性抗体。 靶向GPCRs的治疗性抗体在癌症治疗中具有巨大的潜力 由于它们相对于小分子具有亲和力高、特异性好、毒性低等特点。然而， GPCRs治疗性抗体的发现极具挑战性，因为它们固有的低稳定性、低 在其自然膜环境外的表达和构象异质性。事实上，只有1个 到目前为止，GPCR靶向抗体已经被批准，而且只在日本获得批准。为了克服前车之鉴 提到障碍，Abilita Bio通过生成启用的膜提供了一种创新的解决方案 蛋白质(EMPS™)，代表天然受体的结构和功能增强版本，具有 显著提高了表达能力和热稳定性。高效表达、稳定和 功能折叠的EMPs将能够使用体外抗体分离新的治疗性抗体 显示方法。 所需资金用于：(A)为提议的目标生成突变基因库，并选择增强的 使用我们的定向进化系统的变体；(B)表征表达式、函数和 提高哺乳动物细胞的稳定性；(C)利用进化的EMP作为抗原开发生物扫描分析工具 筛选噬菌体展示抗体库用于初步抗体的分离 并在第二阶段开发。

Project Summary/Abstract Abilita Bio (AB), an innovation driven biotechnology company, requests SBIR Phase I funding for the discovery of novel therapeutic antibodies targeting G protein-coupled receptors (GPCRs) for the treatment of metastatic breast cancer. In cancer therapy each treatment modality has its own limitations. Clinical studies clearly indicate that immunotherapy with checkpoint inhibitors (PD-1/PD-L1, CTLA-4 etc.) can achieve a persistent immune response with improved clinical outcomes. However, this approach is especially effective when administered in combination therapy with targeted therapy aiming at essential components of tumor spreading, homing and growth especially in metastatic cancer. Metastasis is the major factor limiting survival in cancer patients. Therefore, interfering with the molecular mechanisms controlling the metastatic behavior of tumor cells is essential for successfully treating cancer. Experimental and clinical evidence supports the notion that one of the most important mechanisms operating in metastasis involves homeostatic chemokines and their receptors. Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumor cell migration from the primary tumor site to the metastatic homing sites shares many similarities with leukocyte trafficking, which is critically regulated by chemokine-binding GPCRs. Interestingly, several studies have established that prostaglandin E2 (PGE2) secretion by breast cancer cells can upregulate CCR7 expression via activation of prostaglandin-binding GPCRs, EP2 and EP4. This proposal focuses on targeting key GPCRs critically involved in tumor cells metastatic migration and organ-specific invasion using therapeutic antibodies. The use of therapeutic antibodies to target GPCRs has a tremendous potential for the treatment of cancer because of their high affinity, exquisite specificity and low toxicity relative to small molecules. However, GPCRs therapeutic antibody discovery is extremely challenging because of their inherent low stability, low expression and conformational heterogeneity outside their natural membrane environment. In fact, only 1 GPCR-targeting antibody has been approved to date, and only in Japan. In order to overcome the afore mentioned barriers, Abilita Bio offers an innovative solution through the generation of Enabled Membrane Proteins (EMPs™), representing structurally and functionally enhanced versions of natural receptors with significant improved expression and thermostability. The availability of highly expressing, stable and functionally folded EMPs will enable the isolation of novel therapeutic antibodies using in vitro antibody display methods. Funds are requested to (a) generate mutational gene libraries for proposed targets and select for enhanced variants using our directed evolution system; (b) characterize variants for expression, function and enhanced stability in mammalian cells; (c) develop biopanning assay tools using evolved EMPs as antigens and screen phage display antibody libraries for the isolation of preliminary antibody leads to be optimized and developed in Phase II.