GPRC5D protein expression in patients with RRMM and evaluation of TCB potency in vitro. (A) GPRC5D receptor density on MMPCs in bone marrow aspirates from 61 patients with RRMM enrolled in study NCT04557150 (3 samples from 64 could not be reported due to technical issues); these values are depicted on the y-axis of the plot and each dot represents 1 sample; dashed lines on the graph represent mean values for GPRC5D receptor density for 7 cell lines with names of the cell lines specified respectively; GPRC5D receptor density was determined by bead quantification using MESF beads for patient samples and Quantum Simply Cellular kit for cell lines (technical details are described in supplemental Materials and methods). (B) Association between overall cytogenetic risk or individual chromosomal aberrations and GPRC5D binding sites on MM cells in BM; high risk was defined as having ≥1 of the following chromosomal aberrations (regardless of 1q21gain/ampl): del(17p), t(4;14), t(14;16); the 1q21gain/ampl group was defined as having only 1q21gain/ampl reported; N indicates the number of samples that could be evaluated for the target expression. (C-G) MM cell lines with different GPRC5D and BCMA receptor densities (RDs) were cocultured with human peripheral blood mononuclear cells (PBMCs) from 4 to 5 individual healthy donors at an effector-to-target (E:T) ratio of 5:1 and treated with increasing concentrations of forimtamig, 2+1 BCMA TCB, 1+1 GPRC5D TCB, or a untargeted TCB control; LDH release was measured as a surrogate for tumor cell lysis after 40 hours; Triton X-100–treated samples were used to determined maximum tumor lysis and untreated samples to determine spontaneous tumor lysis; expression levels of CD69 and CD25 were measured by flow cytometry as a surrogate for CD8 T-cell activation after 40 hours; EC50 values were calculated from dose-response curves from individual donors; in case curve fits did not allow valid EC50 calculations (if curves either did not reach the top or bottom plateau or had a very wide confidence interval), data were not plotted; and statistical differences between treatment groups were determined per cell line using 1-way analysis of variance (ANOVA) with Tukey posttest analysis. ∗P < .05; ∗∗P < .005. EC50, median effective concentration; LDH, lactate dehydrogenase; MESF, molecules of equivalent soluble fluorophore; ns, not significant.

GPRC5D protein expression in patients with RRMM and evaluation of TCB potency in vitro. (A) GPRC5D receptor density on MMPCs in bone marrow aspirates from 61 patients with RRMM enrolled in study NCT04557150 (3 samples from 64 could not be reported due to technical issues); these values are depicted on the y-axis of the plot and each dot represents 1 sample; dashed lines on the graph represent mean values for GPRC5D receptor density for 7 cell lines with names of the cell lines specified respectively; GPRC5D receptor density was determined by bead quantification using MESF beads for patient samples and Quantum Simply Cellular kit for cell lines (technical details are described in supplemental Materials and methods). (B) Association between overall cytogenetic risk or individual chromosomal aberrations and GPRC5D binding sites on MM cells in BM; high risk was defined as having ≥1 of the following chromosomal aberrations (regardless of 1q21gain/ampl): del(17p), t(4;14), t(14;16); the 1q21gain/ampl group was defined as having only 1q21gain/ampl reported; N indicates the number of samples that could be evaluated for the target expression. (C-G) MM cell lines with different GPRC5D and BCMA receptor densities (RDs) were cocultured with human peripheral blood mononuclear cells (PBMCs) from 4 to 5 individual healthy donors at an effector-to-target (E:T) ratio of 5:1 and treated with increasing concentrations of forimtamig, 2+1 BCMA TCB, 1+1 GPRC5D TCB, or a untargeted TCB control; LDH release was measured as a surrogate for tumor cell lysis after 40 hours; Triton X-100–treated samples were used to determined maximum tumor lysis and untreated samples to determine spontaneous tumor lysis; expression levels of CD69 and CD25 were measured by flow cytometry as a surrogate for CD8 T-cell activation after 40 hours; EC50 values were calculated from dose-response curves from individual donors; in case curve fits did not allow valid EC50 calculations (if curves either did not reach the top or bottom plateau or had a very wide confidence interval), data were not plotted; and statistical differences between treatment groups were determined per cell line using 1-way analysis of variance (ANOVA) with Tukey posttest analysis. ∗P < .05; ∗∗P < .005. EC50, median effective concentration; LDH, lactate dehydrogenase; MESF, molecules of equivalent soluble fluorophore; ns, not significant.

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