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. Author manuscript; available in PMC: 2023 Jan 23.
Published in final edited form as: Sci Transl Med. 2021 Dec 8;13(623):eabh1962. doi: 10.1126/scitranslmed.abh1962

Figure 2. CD38-CCR engagement results in enhanced cytotoxicity of CAR T cells by increasing functional avidity.

Figure 2.

(A) Lysis of luciferase expressing U266 cells (BCMA+CD38−) was measured in a 16 hour BLI assay (n=4 per group) at the indicated E:T ratios. (B) A first generation BCMA-CAR was combined with the CD38Δ construct lacking the intracellular costimulatory tail. BCMA-ζ+CD38Δ T cells and the BCMA-ζ+CD38-28BB T cells were incubated with MM1.S cells and lysis was determined in a 16 hour BLI assay (n=4) at the indicated E:T ratios. Statistical analysis was performed with a two-way ANOVA and subsequent multiple comparison, corrected by Turkey test. ****p<0.0001. (C) MFI of AKT1 phosphorylation (pAKT1) is shown in BCMA-CAR+CD38-CCR T cells activated by stimulation with UM9 cells for 15 minutes as assessed by flow cytometry (n=4 per group). Statistical analysis was performed by one-way ANOVA and subsequent multiple comparison, corrected by Turkey test. *p<0.05, **p<0.01. (D) Granzyme B secretion by CAR T cells co-cultured with ΜΜ1.S (E:T ratio 1:1) for 16 hours is shown (n=4 per group). Statistical analysis was performed by paired t-test. *p<0.05, **p<0.01, ***p<0.001. (E) The strength of interaction between single- and double-targeting T cells and MM1.S target cells was measured. Percentage of total CAR T cells remaining bound to target cells as the acoustic force ramp is applied from 0 to 1000 pN are shown. The dotted line refers to the threshold beyond which all measurement are considered specific binding. (F) The percentage of CAR T cells remaining bound to MM1.S target cells at 1000 pN is shown; data are presented as mean ±SEM from 6 pooled measurements; p-values were calculated using a paired t-test. **p<0.01, ***p<0.001. (G) Affinity characteristics are shown for anti-CD38 antibodies used to generate scFvs. The surface plasmon resonance–determined dissociation constant (KD value, nmol/L) and half-effective concentration (EC50) when titrated on CHO-CD38 cells (μg/mL), described in (29). (H) MM1.S cells were co-cultured at a 1:1 ratio with BCMA-CAR T cells co-expressing CD38-CCRs with gradually lower affinities for CD38 or CD38Δ. Tumor cell lysis was measured in a BLI assay (n=4 per group). Statistical analysis was performed with a paired t-test. (I) Cell supernatants from (H) were harvested to measure granzyme B secretion by ELISA (n=4 per group). Statistical analysis was performed with a paired t-test. ***p<0.001. (J) The strength of interaction between BCMA-ζ + CD38-28BB (EC50 0.3) or BCMA-ζ+CD38A1-28BB (EC50 2.7) T cells and MM1.S target cells is shown as the percentage of total CAR T cells remaining bound to target cells as the acoustic force ramp is applied from 0 to 1000 pN. Dotted line refers to the threshold beyond which all measurement are considered specific binding. (K) The percentage of CAR T cells remaining bound to MM1.S target cells at 1000 pN is shown; data are presented as mean ±SEM from 5 pooled measurements and p-values were calculated using a paired t-test. *p<0.05.