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 journalArticlepeer-review

17 Scopus citations


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
Issue number8
StatePublished - Aug 5 2019

Bibliographical note

Funding Information:
The research study was funded by Aleta Biotherapeutics.

Publisher Copyright:
© 2019 American Chemical Society.


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


Dive into the research topics of 'Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins'. Together they form a unique fingerprint.

Cite this