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 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 Immunology

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 2

CART123 exerts potent antileukemia activity in vitro and in vivo.

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CART123 exerts potent antileukemia activity in vitro and in vivo. (A) CA...
(A) CART123 and CART19 cells were cocultured with B-ALL blasts (the cell line NALM6 or primary blasts, CD19+, CD123+) or a CD19-negative cell line (the CD123+ AML cell line MOLM-14) for 4 to 6 hours. Both CART123 and CART19 showed similar expression of CD107a, a marker of degranulation/activation when cocultured with NALM6 and primary ALL, while only CART123 recognized the CD123+ and CD19– cell line MOLM-14. (B) Cytotoxicity at 24 hours of CART123, CART19, or control T cells (UTD) when cocultured at different E:T ratios with NALM6. Increasing concentration of either CART123 or CART19 led to similar levels of killing of NALM6, while no killing was observed in the control group (UTD). (C) CFSE-stained CART123, CART19, or UTD was cocultured with medium only (TCM), PMA-ionomycin (nonspecific stimulus), MOLM-14 (CD123+, CD19–), or B-ALL blasts (NALM6 or primary blasts, CD19+, CD123+) for 5 days at a 1:1 E:T ratio; then CFSE dilution was analyzed by flow cytometry. High proliferation was observed in both CART123 and CART19 when cocultured with B-ALL blasts, but not in controls (UTD). As a control, CART123 also proliferated with MOLM-14. TCM, tissue culture medium. (D) Cytokine production by CART123, CART19, or UTD incubated with NALM6 for 3 days at 1:1 E:T ratio was analyzed in the culture supernatants. Significant production of several cytokines is noted in CART 19 and CART 123 groups, but not in UTD. (E) Luciferase-positive primary B-ALL blasts (patient UPN#11) were injected in NSG mice, and after 14 days, mice were randomized based on tumor burden to receive either CART123, CART19, or control T cells (UTD). Mice receiving CART123 or CART19, but not UTD, showed quick leukemia remission that was maintained in the long term. All graphs are representative of at least 2 independent experiments. 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. When multiple groups at multiple time points/ratio were compared, Student’s t test or ANOVA for each time point/ratios was used. *P < 0.05; **P < 0.01.