Department of Chemical Engineering and Materials Science , University of Minnesota Twin Cities , 421 Washington Avenue SE , Minneapolis , Minnesota 55455 , United States.
Aleta Biotherapeutics , 27 Strathmore Road , Natick , Massachusetts 01760 , United States.
Mol Pharm. 2019 Aug 5;16(8):3544-3558. doi: 10.1021/acs.molpharmaceut.9b00418. Epub 2019 Jul 15.
CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2 CD19 tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.
CD19 靶向嵌合抗原受体 (CAR) T 细胞 (CAR19s) 在治疗复发/难治性急性淋巴细胞白血病和非霍奇金淋巴瘤方面显示出显著的疗效。然而，使用 CAR T 细胞疗法治疗 CD19 阴性血液系统癌症和实体瘤一直具有挑战性。我们提出了 CD19 融合蛋白 (CD19-FPs)，以利用 CAR19s 的优势，同时将这种经过验证的细胞疗法重新靶向到替代的肿瘤抗原。我们证明了将 CD19 细胞外结构域 (ECD) 和人表皮生长因子受体 2 (HER2) 单链抗体片段融合的能力，以重新靶向 CAR19s 杀死 HER2 CD19 肿瘤细胞。为了增强这项技术的模块化，我们通过酵母展示和流式细胞术选择进行深度突变扫描，对 CD19 ECD 进行了工程改造，以提高蛋白酶抗性和抗 CD19 抗体结合能力。这些增强的 CD19 ECD 显著增加了融合蛋白的表达，在某些情况下甚至恢复了表达，同时保持了靶抗原的亲和力。重要的是，CD19-FPs 将 CAR19s 重新靶向到表达多种不同抗原的肿瘤细胞，包括 HER2、CD20、EGFR、BCMA 和 Clec12A，作为 N 端或 C 端融合，并与抗体片段和纤维连接蛋白配体相连。这项研究提供了对 CD19 序列-功能关系的基本了解，并定义了一个灵活的模块化平台，将 CAR19s 重新靶向到任何肿瘤抗原。
