IL-18-secreting multiantigen targeting CAR T cells eliminate antigen-low myeloma in an immunocompetent mouse model

Abstract

Multiple myeloma is a plasma cell malignancy that is currently incurable with conventional therapies. Following the success of CD19-targeted chimeric antigen receptor (CAR) T cells in leukemia and lymphoma, CAR T cells targeting B-cell maturation antigen (BCMA) more recently demonstrated impressive activity in relapsed and refractory myeloma patients. However, BCMA-directed therapy can fail due to weak expression of BCMA on myeloma cells, suggesting that novel approaches to better address this antigen-low disease may improve patient outcomes. We hypothesized that engineered secretion of the proinflammatory cytokine interleukin-18 (IL-18) and multiantigen targeting could improve CAR T-cell activity against BCMA-low myeloma. In a syngeneic murine model of myeloma, CAR T cells targeting the myeloma-associated antigens BCMA and B-cell activating factor receptor (BAFF-R) failed to eliminate myeloma when these antigens were weakly expressed, whereas IL-18-secreting CAR T cells targeting these antigens promoted myeloma clearance. IL-18-secreting CAR T cells developed an effector-like T-cell phenotype, promoted interferon-gamma production, reprogrammed the myeloma bone marrow microenvironment through type-I/II interferon signaling, and activated macrophages to mediate antimyeloma activity. Simultaneous targeting of weakly-expressed BCMA and BAFF-R with dual-CAR T cells enhanced T-cell:target-cell avidity, increased overall CAR signal strength, and stimulated antimyeloma activity. Dual-antigen targeting augmented CAR T-cell secretion of engineered IL-18 and facilitated elimination of larger myeloma burdens in vivo. Our results demonstrate that combination of engineered IL-18 secretion and multiantigen targeting can eliminate myeloma with weak antigen expression through distinct mechanisms.

Graphical abstract

Figure 1.

Myeloma–directed CAR T cells are impaired by low antigen expression in vivo. (A) Schematic of the APRIL-CAR targeting both BCMA/TACI and scFv–based BCMA and BAFF-R CARs targeting BCMA and BAFF-R, respectively, on a myeloma cell. All vector maps for CARs used in this figure are depicted below. (B) Endogenous expression of TACI, BCMA, and BAFF-R on MOPC315.BM (top, red). Validation of TACI KO with CRISPR-Cas9-gRNA electrporation (purple), and overexpression of a FLAG–tagged murine BCMA that was expressed with a transposase (blue). (C) Schematic of syngeneic MOPC315.BM in vivo model. Mice were lymphodepleted with 450 cGy sublethal total body irradiation (SL-TBI) on day 0 and then injected tail IV with MOPC315.BM. CAR T cells were injected 3 days later. (D) Tumor BLI and survival of mice bearing 4E5 MOPC315.BM FLAG-mBCMA (overexpressed) and treated with BCMA-28-1XX T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM FLAG-mBCMA. 2E6 CAR T cells were injected 3 days later. BCMA-CAR T cells were cultured with 1 μM dasatinib for 2 days prior to injection into mice. (E-F) Tumor BLI and survival of mice bearing MOPC315.BM^WT (TACI^highBCMA^lowBAFF-R^low) and treated with APRIL-CAR T cells with varied costimulatory domains. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E5 MOPC315.BM^WT. 2E6 CAR T cells were injected 3 days later. BLI statistics performed with Vardi area under the curve (AUC) analysis and survival statistics with Mantel-Cox log-rank test. Data are combined from at least 4 independent experiments. (G) T-cell BLI of mice treated with CD45.1⁺ APRIL-CAR T cells labeled with gLuc-GFP. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E5 MOPC315.BM^WT. 2E6 CAR T cells were injected 3 days later. T cells were imaged weekly starting on day 6. Statistics were performed with the 1-way analysis of variance (ANOVA) test on day 27. Data are combined from at least 2 independent experiments. (H) CD45.1⁺ T-cell abundance measured in the peripheral blood of mice from panel G on day 29, as measured by flow cytometry. Statistics were performed with the 1-way ANOVA test. Data are representative of at least 2 independent experiments. (I-J) Tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with APRIL, BCMA, or BAFF-R-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM^low. 2E6 CAR T cells were injected 3 days later. ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 2.

Engineered IL-18 secretion by CAR T cells improves therapeutic activity against antigen-high and antigen-low myeloma. (A) Schematic of APRIL, BCMA, or BAFF-R CAR T cells engineered to secrete IL-18. Vector maps for CAR constructs used in this figure are depicted to the right. (B) Engineered IL-18 secretion levels by APRIL-CAR T cells ± engineered IL-18 secretion. Approximately 100 000 CAR T cells were cultured for 24 hours in IL-2 and the supernatant was collected for ELISA. Concentration was determined based on standard curve measurements. Statistical analysis performed with the Student t test. (C) IFN-γ secretion by APRIL-CAR T cells ± engineered IL-18 secretion. Supernatant from setup in panel B was analyzed using an IFN-γ flow cytometric bead array kit. Statistical analysis performed with the Student t test. (D) Tumor BLI and survival of mice bearing MOPC315.BM^WT and treated with CD28-costimulated, IL-18-secreting APRIL-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E5 MOPC315.BM^WT. 2E6 CAR T cells were injected 3 days later. Data are combined from at least 2 independent experiments. (E) Tumor BLI and survival of mice bearing MOPC315.BM^WT and treated with CD28-4-1BB-costimulated IL-18-secreting APRIL-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E5 MOPC315.BM^WT. 2E6 CAR T cells were injected 3 days later. (F) Schematic of syngeneic antigen-low MOPC315.BM^low in vivo model (used in panels G-I). Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM^low. 2E6 CAR T cells were injected 3 days later. (G) Tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with IL-18-secreting APRIL or BCMA-CAR T cells. Tumor BLI was performed weekly starting on day 7. Data are combined from at least 2 independent experiments. (H) Tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with BAFF-R-28-1XX CAR T cells ± IL-18. Tumor BLI was performed weekly starting on day 7. (I) Tumor BLI, T-cell BLI, and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with APRIL-BB-1XX/IL-18 T cells compared with control APRIL-BB-1XX T cells without engineered IL-18 expression. T-cell BLI was performed weekly starting on day 6. Tumor BLI was performed weekly starting on day 7. Data are combined from at least 2 independent experiments. (J) Survival of APRIL-BB-1XX/IL-18 treated complete responder mice that were rechallenged with 4E5 MOPC315.BM TACI^KO(BCMA^lowBAFF-R^low) 93 days after original experiment start date. Mice were not irradiated before re-challenge. Statistical analysis of all survival curves was performed with the Mantel-Cox log-rank test. ELISA, enzyme-linked immunosorbent assay. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.

Figure 3.

IL-18-secreting CAR T cells increase interferon signaling and drive effector-like differentiation of T cells in vivo. (A) Experimental setup describing in vivo antigen-low IL-18-CAR T-cell model and BM harvest for flow or single-cell sequencing. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM^low. 2E6 CAR T cells were injected 3 days later. Harvest data for single-cell sequence or flow cytometry were collected on day 7 of experiment unless otherwise specified. (B) UMAP projection of 34628 CD45⁺ cells post-QC processing, annotated by different cell types (left) and biological group (right). (C) GSEA plot depicting enrichment of the type-I/II interferon signaling pathways in T cells from APRIL-BB-1XX/IL-18 treated mice. (D-E) Dotplot of selected CITE-seq epitope expression (D) and RNA transcript expression (E) related to antigen presentation, interferon signaling/response, T-cell activation/effector function, T-cell stemness, and cell cycle. Columns represent 3 replicate mice in each group. (F) Abundance of total T cells, CD45.2⁺ host T cells, and CD45.1⁺ donor CAR T cells, as measured by flow cytometry. Data are combined from at least 2 independent experiments. Statistics performed with the 1-way ANOVA test. (G) Expression of stemness/naïve-like markers in CD45.1⁺ donor CAR T cells, as measured by flow cytometry. Data are combined from at least 2 independent experiments. (H) UMAP projection of T cells and annotation of CD45.2⁺ host T cells and CD45.2^– donor CAR T cells. Annotations were assigned using CD45.2 expression with CITE-seq. (I) Volcano plots of the Wilcoxon rank-sum scores from the differential gene expression analysis of APRIL-BB-1XX/IL-18 vs APRIL-BB-1XX CAR T-cell treatment in CD45.2⁺ host T cells and CD45.2^– donor CAR T cells, respectively. Genes with adjusted P value = 1 were filtered out before plotting. (J) GSEA plot displaying upregulation of NF-κB signaling pathway in CD45.2^– donor APRIL-BB-1XX/IL-18 T cells. UMAP: Uniform Manufold Approximation and Projection; GSEA: gene set enrichment analysis. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 4.

IL-18-mediated IFN-γ secretion activates macrophages to promote elimination of antigen-low myeloma. (A) Z-score heat map of proinflammatory cytokines in the peripheral blood of CAR T-cell–treated mice on day 8 of experiment, measured with the BioLegend Legendplex mouse inflammation panel of 13 cytokines. (B) IFN-γ concentration in the peripheral blood from panel A, measured with the BioLegend Legendplex mouse inflammation kit. Statistics were performed with the 1-way ANOVA test. (C) Abundance of CD11b⁺ F4/80⁺ macrophages in the BM of CAR T-cell–treated mice. Data are combined from at least 2 independent experiments. (D) Volcano plot of selected upregulated genes in BM macrophages from APRIL-BB-1XX/IL-18 vs APRIL-BB-1XX T-cell–treated mice. Genes with P value = 1 were filtered out before plotting. (E) GSEA plots of upregulated pathways related to hallmark interferon-gamma response, antigen presentation/processing and glucose utilization in BM macrophages from APRIL-BB-1XX/IL-18 treated mice. (F) Dotplot of mRNA transcript expression of genes related to antigen presentation and interferon response (more M1-like), M2/immunosuppression, macrophage activation, and glucose utilization in BM macrophages from mice treated with APRIL-BB-1XX T cells ± IL-18. Columns represent 3 replicate mice in each group. (G-H) Expression of MHC-II in BM macrophages as measured by CITE-seq (G) and flow cytometry (H). Flow cytometry data are combined from at least 2 independent experiments. (I) Production of tumor necrosis factor α (TNF-α) in BM macrophages as measured with flow cytometry, taken from a BM aspirate on day 10 of experiment. (J) Experimental setup of macrophage culture and isolation, followed by coculture of macrophages with T cells and antigen-low MOPC315.BM^low. BM was cultured with 25 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then macrophages were harvested. Approximately 10 000 macrophages were cultured with 10 000 MOPC315.BM^low and 100 000 CAR T cells. (K-L) Incucyte cytotoxicity assay of APRIL-CAR T cells ± IL-18 expression and MOPC315.BM^low cocultured with macrophages. AUC was generated and statistical analysis was performed with the 1-way ANOVA test. Results are depicted as comparison of each T-cell group with macrophage coculture, or macrophages alone (K) and each T-cell product ± macrophages (L). Data are representative of at least 2 independent experiments. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 4.

IL-18-mediated IFN-γ secretion activates macrophages to promote elimination of antigen-low myeloma. (A) Z-score heat map of proinflammatory cytokines in the peripheral blood of CAR T-cell–treated mice on day 8 of experiment, measured with the BioLegend Legendplex mouse inflammation panel of 13 cytokines. (B) IFN-γ concentration in the peripheral blood from panel A, measured with the BioLegend Legendplex mouse inflammation kit. Statistics were performed with the 1-way ANOVA test. (C) Abundance of CD11b⁺ F4/80⁺ macrophages in the BM of CAR T-cell–treated mice. Data are combined from at least 2 independent experiments. (D) Volcano plot of selected upregulated genes in BM macrophages from APRIL-BB-1XX/IL-18 vs APRIL-BB-1XX T-cell–treated mice. Genes with P value = 1 were filtered out before plotting. (E) GSEA plots of upregulated pathways related to hallmark interferon-gamma response, antigen presentation/processing and glucose utilization in BM macrophages from APRIL-BB-1XX/IL-18 treated mice. (F) Dotplot of mRNA transcript expression of genes related to antigen presentation and interferon response (more M1-like), M2/immunosuppression, macrophage activation, and glucose utilization in BM macrophages from mice treated with APRIL-BB-1XX T cells ± IL-18. Columns represent 3 replicate mice in each group. (G-H) Expression of MHC-II in BM macrophages as measured by CITE-seq (G) and flow cytometry (H). Flow cytometry data are combined from at least 2 independent experiments. (I) Production of tumor necrosis factor α (TNF-α) in BM macrophages as measured with flow cytometry, taken from a BM aspirate on day 10 of experiment. (J) Experimental setup of macrophage culture and isolation, followed by coculture of macrophages with T cells and antigen-low MOPC315.BM^low. BM was cultured with 25 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then macrophages were harvested. Approximately 10 000 macrophages were cultured with 10 000 MOPC315.BM^low and 100 000 CAR T cells. (K-L) Incucyte cytotoxicity assay of APRIL-CAR T cells ± IL-18 expression and MOPC315.BM^low cocultured with macrophages. AUC was generated and statistical analysis was performed with the 1-way ANOVA test. Results are depicted as comparison of each T-cell group with macrophage coculture, or macrophages alone (K) and each T-cell product ± macrophages (L). Data are representative of at least 2 independent experiments. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 5.

BAFF-R/APRIL dual-CAR T cells with engineered IL-18 secretion eliminate antigen-low myeloma in a stress-dose model. (A) Schematic of BAFF-R/APRIL dual-CAR T cells and the corresponding CAR vector maps. (B) Incucyte cytotoxicity assay of BAFF-R/APRIL dual or single-CAR T cells vs 10 000 IRFP713⁺ MOPC315.BM^low. Near-infrared signal was measured over time with the Incucyte SX5. Statistical analysis was performed with the 1-way ANOVA test at 72-hour end point. Data are representative of at least 2 independent experiments. (C) Binding avidity force between MOPC315.BM^low and BAFF-R/APRIL dual or single-CAR T cells. Myeloma cells were seeded on poly-L-lysine coated plates before adding EGFP⁺ T cells. Lumicks C-trap optical tweezers were used to locate EGFP⁺ T cells, bring them in contact with myeloma cells, and then separate them, allowing separating force to be recorded. Statistical analysis was performed with the 1-way ANOVA test. Data are combined from at least 2 independent experiments. (D) Confocal microscopy of coexpressed BAFF-R-EGFP and APRIL-tagRFP CARs present at the synapse interface between CAR T cells and MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low). CAR T cells were cocultured with MOPC315.BM^low for 2 hours and then fixed and processed for imaging. Z-stack images were acquired with Leica Stellaris 8 at 63× magnification and room temperature (25°C), in the following channels: green, red, and DAPI. Data were analyzed on the Imaris software (Oxford Instruments) and 2D images containing a clear representation of the T-cell:MOPC315.BM^low synaptic interface were exported. (E) NFAT signaling of BAFF-R/APRIL dual or single-CAR T cells when exposed MOPC315.BM^WT or MOPC315.BM^low myeloma cells. A total of 20 000 CAR T cells containing an NFAT-EGFP response element were cocultured with 60 000 myeloma cells for 24 hours. NFAT-GFP signaling was measured with flow cytometry. Data are representative of at least 2 independent experiments. (F) NF-κB signaling of BAFF-R/APRIL dual or single-CAR T cells. 10 000 CAR T cells containing an NF-κB-EGFP response element were cocultured with 30 000 MOPC315.BM^low cells. NF-κB-EGFP signal was measured over time with the Incucyte SX5. Statistical analysis was performed with the 1-way ANOVA test at 120 hours. Data are representative of at least 2 independent experiments. (G) Antigen-low myeloma standard dosing schematic, tumor BLI and survival of mice bearing MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with BAFF-R/APRIL dual or single APRIL or BAFF-R-CAR T cells. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 4E5 MOPC315.BM^low. 2E6 CAR T cells were injected 3 days later. Statistical analysis of survival was performed with the Mantle-Cox log-rank test. Data are combined from at least 2 independent experiments. (H) Schematic of IL-18-secreting BAFF-R/APRIL dual-CAR T cells and the corresponding vector maps. (I) Model setup of stress-dose model of MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) with a 5× increase in tumor dose and 20× decrease in T-cell dose. Mice were lymphodepleted with 450 cGy SL-TBI on day 0 and then injected tail IV with 2E6 MOPC315.BM^low. 1E5 CAR T cells were injected 3 days later. (J) Tumor BLI and survival of mice bearing 2E6 MOPC315.BM^low (TACI^KOBCMA^lowBAFF-R^low) and treated with a low dose of 1E5 IL-18-secreting BAFF-R/APRIL dual-CAR T cells, BAFF-R/APRIL dual-CAR T cells alone, BAFF-R/IL-18 CAR T cells, or APRIL/IL-18 CAR T cells. Statistical analysis of survival was performed with the Mantel-Cox log-rank test. Data are combined from at least 3 independent experiments. (K) ELISA of IL-18 production from BAFF-R-28-1XX/APRIL-BB-1XX/IL-18 compared with BAFF-R-28-1XX/IL-18 and APRIL-BB-1XX/IL-18 CAR T cells when cocultured with MOPC315.BM^low. Statistical analysis was performed with the 1-way ANOVA test. DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.