Figure 1.
BCMA CAR design and optimization. (A) Schematic of CAR constructs containing the C11 scFv in VH/VL or VL/VH configuration (HL or LH), 4-1BB, or CD28 costimulatory domain (costim), CD3ζ signaling domain, T2A ribosomal skip sequence, and EGFRt. The previously described BCMA-2 CAR40 is also depicted, and contains a CD8α hinge and transmembrane (TM) domain, and a CD28 costimulatory domain. (B) CD8+ T cells were transduced with C11/HL and C11/LH 4-1BB/CD3ζ CARs containing a long spacer sequence (IgG4 hinge-CH2-CH3NQ) and selected for EGFRt expression. IFN-γ production in supernatants of CAR T cells after stimulation with K562, K562/BCMA, and 8226 cells for 24 hours was measured by ELISA. T cells were prepared from 2 different donors and values were normalized to production of C11/LH/4-1BB/CD3ζ against K562/BCMA. (C) Schematic of various spacers used in the C11/HL/4-1BB/CD3ζ BCMA-CARs. (D) IFN-γ production in supernatants of CD8+ BCMA CAR T cells with the indicated spacers after stimulation with 8226 and U266 myeloma cell lines for 24 hours as measured by ELISA. IFN-γ was normalized to production of long spacer CAR against 8226 or U266, respectively. (E) IFN-γ (left) and IL-2 (right) in supernatants of CD4+ and CD8+ T cells expressing C11/HL/3ST/4-1BB/CD3ζ, C11/HL/3ST/CD28/CD3ζ, or BCMA-2 CARs after 24 hours coculture with the indicated BCMA− and BCMA+ target cells. Values were normalized to BCMA-2 against K562/BCMA. (F) Proliferation of CD4+ and CD8+ BCMA CAR T cells after stimulation with target cells for 72 hours analyzed by CFSE dilution assay. (G) Representative CAR expression on CD8+ BCMA CAR T cells after isolation and expansion as measured by BCMA/Fc conjugated to APC. The E:T ratio was 2:1 in all assays. Data in panels D through G are summarized (D-E) or representative (F-G) of 2 or more individual experiments with T cells prepared from different donors. Data depicted in bar graphs represent mean plus standard error of the mean (SEM). ***P < .001; as determined by 1-way ANOVA with the Dunnett posttest. ns, not significant.

BCMA CAR design and optimization. (A) Schematic of CAR constructs containing the C11 scFv in VH/VL or VL/VH configuration (HL or LH), 4-1BB, or CD28 costimulatory domain (costim), CD3ζ signaling domain, T2A ribosomal skip sequence, and EGFRt. The previously described BCMA-2 CAR40  is also depicted, and contains a CD8α hinge and transmembrane (TM) domain, and a CD28 costimulatory domain. (B) CD8+ T cells were transduced with C11/HL and C11/LH 4-1BB/CD3ζ CARs containing a long spacer sequence (IgG4 hinge-CH2-CH3NQ) and selected for EGFRt expression. IFN-γ production in supernatants of CAR T cells after stimulation with K562, K562/BCMA, and 8226 cells for 24 hours was measured by ELISA. T cells were prepared from 2 different donors and values were normalized to production of C11/LH/4-1BB/CD3ζ against K562/BCMA. (C) Schematic of various spacers used in the C11/HL/4-1BB/CD3ζ BCMA-CARs. (D) IFN-γ production in supernatants of CD8+ BCMA CAR T cells with the indicated spacers after stimulation with 8226 and U266 myeloma cell lines for 24 hours as measured by ELISA. IFN-γ was normalized to production of long spacer CAR against 8226 or U266, respectively. (E) IFN-γ (left) and IL-2 (right) in supernatants of CD4+ and CD8+ T cells expressing C11/HL/3ST/4-1BB/CD3ζ, C11/HL/3ST/CD28/CD3ζ, or BCMA-2 CARs after 24 hours coculture with the indicated BCMA− and BCMA+ target cells. Values were normalized to BCMA-2 against K562/BCMA. (F) Proliferation of CD4+ and CD8+ BCMA CAR T cells after stimulation with target cells for 72 hours analyzed by CFSE dilution assay. (G) Representative CAR expression on CD8+ BCMA CAR T cells after isolation and expansion as measured by BCMA/Fc conjugated to APC. The E:T ratio was 2:1 in all assays. Data in panels D through G are summarized (D-E) or representative (F-G) of 2 or more individual experiments with T cells prepared from different donors. Data depicted in bar graphs represent mean plus standard error of the mean (SEM). ***P < .001; as determined by 1-way ANOVA with the Dunnett posttest. ns, not significant.

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