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Immunotherapy

High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity

Leukemia volume 30, pages 1355–1364 (2016)Cite this article

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Abstract

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

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Acknowledgements

This work was supported by grants from the NIH (RO1-CA168900; to DJP) and (T32-AI070099; to RCL) and by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health (to DSD). We thank Gwenn Danet-Desnoyers, Tony Secreto, Josh Glover and Winifred Trotman at the SCXC for their assistance with leukemia and BM acquisition and xenograft services. Bioluminescent imaging was supported by the University of Pennsylvania small animal imaging core facility.

Author contributions

RCL and DJP designed experiments and wrote the paper. RCL conducted experiments, analyzed data and prepared the figures. YF and DSD isolated the scFvs and prepared and conducted experiments with soluble FRβ scFvs and IgGs. KS prepared mRNA CAR reagents. MP conducted in vivo imaging. AK scored the CFU. All authors have read and approved the submitted manuscript.

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Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    R C Lynn, K Schutsky, M Poussin & D J Powell Jr

  2. Protein Interactions Section, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA

    Y Feng & D S Dimitrov

  3. Division of Hematology and Oncology, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    A Kalota

  4. Department of Pathology and Laboratory Medicine, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    D J Powell Jr

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  1. R C Lynn
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Correspondence to D J Powell Jr.

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Lynn, R., Feng, Y., Schutsky, K. et al. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity. Leukemia 30, 1355–1364 (2016). https://doi.org/10.1038/leu.2016.35

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