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

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

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3544-3558
Number of pages15
JournalMolecular pharmaceutics
Volume16
Issue number8
DOIs
StatePublished - Aug 5 2019

Keywords

  • CD19
  • chimeric antigen receptors
  • deep mutational scanning
  • protein fusion
  • protein solubility
  • yeast surface display

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't

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    Klesmith, J. R., Su, L., Wu, L., Schrack, I. A., Dufort, F. J., Birt, A., Ambrose, C., Hackel, B. J., Lobb, R. R., & Rennert, P. D. (2019). Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins. Molecular pharmaceutics, 16(8), 3544-3558. https://doi.org/10.1021/acs.molpharmaceut.9b00418