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.BMWT (TACIhighBCMAlowBAFF-Rlow) 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.BMWT. 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.BMWT. 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.BMlow (TACIKOBCMAlowBAFF-Rlow) 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.BMlow. 2E6 CAR T cells were injected 3 days later. ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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.BMWT (TACIhighBCMAlowBAFF-Rlow) 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.BMWT. 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.BMWT. 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.BMlow (TACIKOBCMAlowBAFF-Rlow) 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.BMlow. 2E6 CAR T cells were injected 3 days later. ns, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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