Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins

• DOI: 10.1021/acs.molpharmaceut.9b00418
• Publication Date: 2019-06-26T22:45:45Z
• Authors: Justin R. Klesmith; Lihe Su; Lan Wu; Ian A. Schrack; Fay J. Dufort; Alyssa Birt; Christine Ambrose; Benjamin J. Hackel; Roy R. Lobb; Paul D. Rennert
• Keywords: 0301 basic medicine; 0303 health sciences; Receptors, Chimeric Antigen; Receptor, ErbB-2; Recombinant Fusion Proteins; T-Lymphocytes; Antigens, CD19; Protein Engineering; Immunotherapy, Adoptive; 3. Good health; 03 medical and health sciences; HEK293 Cells; Protein Domains; Antigens, Neoplasm; Mutagenesis; Cell Line, Tumor; Neoplasms; Humans; Single-Chain Antibodies

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, use CAR T-cell therapy against CD19-negative hematological cancers solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage benefits CAR19s while retargeting this validated cellular alternative tumor antigens. demonstrate ability a fusion CD19 extracellular domain (ECD) human epidermal growth factor 2 (HER2) single-chain antibody fragment retarget kill HER2+ CD19– cells. To enhance modularity technology, we engineered more robust ECD via deep mutational scanning with yeast display flow cytometric selections for improved protease resistance anti-CD19 binding. These enhanced ECDs significantly increase, some cases recover, protein expression maintaining target affinity. Importantly, CD19-FPs cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, Clec12A as N- or C-terminal fusions linked both fragments fibronectin ligands. This study provides fundamental insights into sequence–function relationships defines flexible modular platform any antigen.