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.

Figures

Figure 1. CD123 is highly expressed in B-ALL, including the LIC and the CD19-negative relapses occurring after CD19-targeted immunotherapies.

(A) High expression of CD123 in 42 R/R B-ALL samples (gated on blasts: SSClo, single, live, CD45dim/neg). (B) CD123 and CD19 are typically coexpressed in B-ALL blasts, although minor CD19-negative and CD123-negative blasts can be detected at very low frequencies. (C) CD123 is expressed in the LIC compartment (red), defined as CD34+CD38– blasts (blue represents control CD45++ cells). RIght panel shows expression of CD123 in the ALL LIC of 23 B-ALL patients. (D) B-ALL blasts were sorted using a flow cytometry sorter, based on the expression of CD19 and CD123, obtaining 4 subsets: CD19+CD123–, CD19+CD123+, CD19–CD123–, and CD19–CD123+. These 4 subsets were highly pure and were analyzed by FISH for their specific genetic marker. Boxes indicate percentage of FISH-positive cells in each subset. Importantly, CD19–CD123+ blasts were positive in most cases. (E) The sorting experiment was repeated in a total of 6 B-ALL samples with different FISH abnormalities, and results were consistent. (F) Injection of sorted CD19-negative CD123-positive leukemic subpopulations into NSG mice led to reconstitution of the original B-ALL phenotype (BM, day 120), confirming this population’s LIC function (graph representative of 2 independent experiments each with 2 mice per group). (G) CD123 expression (MFI, mean fluorescence intensity) is maintained in CD19-negative B-ALL relapses occurring after CART19 (CTL019) treatment; representative case is shown. Gating is based on isotype control.

Figure 2. CART123 exerts potent antileukemia activity in vitro and in vivo.

(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.

Figure 3. CART123, but not CART19, can successfully target CD19-negative B-ALL relapses.

(A) A human xenograft model of CD19-negative relapse; experiment schematic. Two groups of NSG mice were respectively engrafted with B-ALL blasts (luciferase positive) originally obtained from the same patient (patient UPN#09) at baseline and when the patient relapsed with a CD19-negative disease after CART19. At day 17, mice in each group were randomized to receive CART19 or control T cells (UTD). Leukemia burden was followed using bioluminescence (BLI). (B) In both groups (baseline and relapse), mice treated with UTD showed disease progression (top graph). CART19 are only capable of inducing responses in the group of mice engrafted with baseline disease, while no antileukemia effect is observed in the group engrafted with CD19-negative relapse disease (bottom graph). (C) In another in vivo model, baseline (CD19+, CBG luciferase+) and relapsed (CD19–, CBR luciferase+) blasts from the same patient were mixed 1:1 and injected in the same NSG mice. Mice were then treated with CART19 or UTD, and the 2 B-ALL populations (baseline vs. relapse) were followed by bioluminescence. In mice treated with UTD, both populations showed progression (CBG and CBR bioluminescence), while in CART19-treated mice, only the relapsed clone (CD19-negative) showed frank progression (CBR). (D) CART123 successfully eliminated CD19-negative B-ALL relapses in vivo. Experiment schematic: luciferase-positive primary CD19-negative relapsed B-ALL blasts were injected in NSG mice, and after 17 days, mice were randomized based on tumor burden to receive either CART123, CART19, control T cells (UTD), or no treatment. (E) Mice receiving CART123 but not CART19 or UTD showed quick leukemia remission that was maintained in the long term (>120 days), leading to a significant advantage in overall survival (F). All graphs arerepresentative of 2 independent experiments (6-8 mice per group). Student’s t test or ANOVA for each time point/ratio was used. Survival curves were compared using the log-rank test. ***P < 0.001; ****P < 0.0001.

Figure 4. Preventing CD19-negative relapses using combination of CART19 and CART123.

(A) Experiment schematic: NSG mice were engrafted with a 1:1 mixture of B-ALL blasts obtained from a patient (patient UPN#09) at baseline when the disease was CD19+CD123+ (CBG luciferase+) and at relapse when the disease was CD19–CD123+ (CBR luciferase+). At day 17, mice were randomized to receive CART19, control T cells (UTD), or the 1:1 pooled combination of CART123 and CART19. (B) Mice treated with UTD showed progression of both CD19+ baseline ALL and CD19– relapsed ALL, while mice receiving CART19 showed progression with a CD19-negative disease. Only CART123 or the combination of CART123 and CART19 was able to prevent the onset of CD19– relapse. (C) Representative leukemia phenotype observed in the spleen of mice sacrificed at day 50 (gated on human SSClo, CD45dim, live, single, GFP+ blasts): UTD-treated mice progress, with both CD19+ and CD19– ALL, while CART19-treated mice showed clear CD19-negative progression. CART123 and the combination of CART123 and CART19 led to the clearance of both CD19+ and CD19– leukemia in most of the mice. Representative of 2 independent experiments. Student’s t test for each time point/ratio was used. *P < 0.05; **P < 0.01.

Figure 5. Potent activation and dual-specific immune synapse formation of dual CART19/123 cells.

(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.

Figure 6. Dual CART19/123 cells are highly effective against B-ALL in vivo.

(A) NSG mice were engrafted with a B-ALL cell line (NALM6, CBG+). At day 7, mice were randomized based on tumor burden (BLI) to receive control T cells (UTD), CART19, CART123, the 1:1 pooled combination of CART123 and CART19, or the dual CART19/123 (same total number of CAR+ cells). The tumor burden 6 days after T cell infusion (day 13) is shown in the graph: the deepest short-term antileukemia response is observed in the dual CART group. Monitoring of tumor burden (BLI) over time shows that only the dual CART19/123-treated mice have long-term CR (P = 0.02 at day 63). (B) Bioluminescence imaging of mice receiving the different treatments at day 7 (before T cell infusion) and day 13 (6 days after T cell infusion). Only mice receiving dual CART19/123 reached a quick and deep complete response. All graphs representative of 2 independent experiments. (C) In a primary B-ALL xenograft model (patient UPN#11), dual CART19/123 have a superior antileukemia activity (shown as reduced bioluminescence) as compared with pooled CART19 + CART123 at early time point (day 9), and this was correlated by a significantly higher T cell engraftment in the PB (D). (E) Tumor burden imaging (as mean bioluminescence) demonstrated a better antileukemia activity of dual CART19/123 as compared with pooled CART19 + CART123 or single CART. Panels B–E 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/ratios were compared, the Student’s t test or ANOVA for each time point/ratio was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.