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

Publisher: American Chemical Society Country of Publication: United States NLM ID: 101197791 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1543-8392 (Electronic) Linking ISSN: 15438384 NLM ISO Abbreviation: Mol Pharm Subsets: MEDLINE

Original Publication: Washington, DC : American Chemical Society, c2004-

Antigens, CD19/*metabolism ; Immunotherapy, Adoptive/*methods ; Neoplasms/*therapy ; Recombinant Fusion Proteins/*metabolism ; Single-Chain Antibodies/*metabolism ; T-Lymphocytes/*immunology; Antigens, CD19/genetics ; Antigens, CD19/immunology ; Antigens, Neoplasm/immunology ; Cell Line, Tumor ; HEK293 Cells ; Humans ; Mutagenesis ; Neoplasms/immunology ; Neoplasms/pathology ; Protein Domains/genetics ; Protein Engineering ; Receptor, ErbB-2/antagonists & inhibitors ; Receptors, Chimeric Antigen/immunology ; Receptors, Chimeric Antigen/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/immunology ; Single-Chain Antibodies/genetics ; Single-Chain Antibodies/immunology ; T-Lymphocytes/metabolism ; T-Lymphocytes/transplantation

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.

Keywords: CD19; chimeric antigen receptors; deep mutational scanning; protein fusion; protein solubility; yeast surface display

0 (Antigens, CD19)
0 (Antigens, Neoplasm)
0 (CD19 molecule, human)
0 (Receptors, Chimeric Antigen)
0 (Recombinant Fusion Proteins)
0 (Single-Chain Antibodies)
EC 2.7.10.1 (ERBB2 protein, human)
EC 2.7.10.1 (Receptor, ErbB-2)

Date Created: 20190627 Date Completed: 20200625 Latest Revision: 20200625

20221213

10.1021/acs.molpharmaceut.9b00418

31242389