Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies
Marco Ruella, … , Stephan A. Grupp, Saar Gill
Marco Ruella, … , Stephan A. Grupp, Saar Gill
Published October 3, 2016; First published August 29, 2016
Citation Information: J Clin Invest. 2016;126(10):3814-3826. https://doi.org/10.1172/JCI87366.
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Research Article Hematology

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies

Abstract

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies

Authors

Marco Ruella, David M. Barrett, Saad S. Kenderian, Olga Shestova, Ted J. Hofmann, Jessica Perazzelli, Michael Klichinsky, Vania Aikawa, Farzana Nazimuddin, Miroslaw Kozlowski, John Scholler, Simon F. Lacey, Jan J. Melenhorst, Jennifer J.D. Morrissette, David A. Christian, Christopher A. Hunter, Michael Kalos, David L. Porter, Carl H. June, Stephan A. Grupp, Saar Gill

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Figure 5

Potent activation and dual-specific immune synapse formation of dual CART19/123 cells.

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Potent activation and dual-specific immune synapse formation of dual CAR...
(A) Dual CART19/123 cells showed CD107a degranulation when cocultured for 6 hours with CD19-positive K562 cell line or CD123+ K562, while single CART19+ or CART123+ cells were only able to recognize CD19+ or CD123+ K562, respectively. (B) A bicistronic P2A plasmid carrying both CART19 and CART123 was used to generate lentivirus and transduce T cells. As shown in the dot plot, transduced T cell display combined expression of CART19 and CART123. (C) Dual and single CAR+ Jurkat NFAT reporter cell lines were generated and used to test NFAT activation dynamics when cocultured with CD19+CD123+ K562 cells. At 1 hour, dual CARTs showed significantly higher NFAT activation as compared with either single-positive CART (CART19 or CART123). (D) Confocal imaging of dual CART revealed that both CARs (CART19, red, CART123, green) are simultaneously engaged in the same immune synapse (yellow) with leukemic cells (NALM6, stained with CellTrace Violet). Panels A–C are representative of 2 independent experiments. Magnification, ×63 (oil immersion). Student’s t test was used to compare 2 groups; in analysis where multiple groups were compared, 1-way ANOVA was performed with Holm-Šidák correction for multiple comparisons. *P < 0.05; **P < 0.01.