Preclinical activity and determinants of response of the GPRC5DxCD3 bispecific antibody talquetamab in multiple myeloma

Abstract

Cell surface expression levels of GPRC5D, an orphan G protein-coupled receptor, are significantly higher on multiple myeloma (MM) cells, compared with normal plasma cells or other immune cells, which renders it a promising target for immunotherapeutic strategies. The novel GPRC5D-targeting T-cell redirecting bispecific antibody, talquetamab, effectively kills GPRC5D+ MM cell lines in the presence of T cells from both healthy donors or heavily pretreated MM patients. In addition, talquetamab has potent anti-MM activity in bone marrow (BM) samples from 45 patients, including those with high-risk cytogenetic aberrations. There was no difference in talquetamab-mediated killing of MM cells from newly diagnosed, daratumumab-naïve relapsed/refractory (median of 3 prior therapies), and daratumumab-refractory (median of 6 prior therapies) MM patients. Tumor cell lysis was accompanied by T-cell activation and degranulation, as well as production of pro-inflammatory cytokines. High levels of GPRC5D and high effector:target ratio were associated with improved talquetamab-mediated lysis of MM cells, whereas an increased proportion of T cells expressing PD-1 or HLA-DR, and elevated regulatory T-cell (Treg) counts were associated with suboptimal killing. In cell line experiments, addition of Tregs to effector cells decreased MM cell lysis. Direct contact with bone marrow stromal cells also impaired the efficacy of talquetamab. Combination therapy with daratumumab or pomalidomide enhanced talquetamab-mediated lysis of primary MM cells in an additive fashion. In conclusion, we show that the GPRC5D-targeting T-cell redirecting bispecific antibody talquetamab is a promising novel antimyeloma agent. These results provide the preclinical rationale for ongoing studies with talquetamab in relapsed/refractory MM.

Graphical abstract

Figure 1.

GPRC5D protein and RNA expression levels in normal and clonal plasma cells. (A) Cell surface expression of GPRC5D, as determined by MFI, on plasma cells was assessed by flow cytometry in BM samples obtained from 27 NDMM, 22 daratumumab-naïve RRMM, and 25 daratumumab-refractory MM patients and in normal BM from 11 age-matched healthy donors. Groups were compared using Mann-Whitney U test. (B) GPRC5D cell surface protein expression on MM cells was compared with the expression on BM-localized immune cells in the same patient’s samples by Wilcoxon matched-pairs test. (C) The same analysis was performed in BM samples obtained from healthy donors (n = 11). (D) Cell surface expression of BCMA and CD38 on plasma cells in BM samples from the same patients and healthy donors. (E) Gene expression profiling was performed on purified CD138⁺ plasma cells, publicly available datasets were used. mRNA expression of GPRC5D was compared between normal plasma cells obtained from healthy donors and clonal plasma cells from MGUS, SMM and NDMM patients. GPRC5D mRNA expression is plotted as Log2 transformed, MAS5 normalized values. (F) In datasets from 5 large trials (HOVON65, TT2, TT3, MRC IX, and APEX, n = 1421), GPRC5D mRNA expression in MM cells was compared between patients with or without specific cytogenetic abnormalities (gain(1q), del(13q), del(17p), and IGH translocations [t(4;14), t(11;14), and t(MAF)]). Groups were compared using Wilcoxon rank-sum test. (A-F) Each dot represents an individual sample, with box and whiskers, representing median, 25th-75th percentile, and range. (G) Univariate Cox regression analysis was performed to evaluate the effect of GPRC5D expression on progression-free survival and overall survival. *P < .05; ***P < .001; ****P < .0001. ns, not significant.

Figure 2.

Talquetamab-mediated lysis of cell lines. (A) GPRC5D protein expression on cell lines was assessed by flow cytometry. Representative overlay histograms depict GPRC5D cell surface expression (red histogram) on 4 luciferase-transduced (LUC⁺) MM cell lines (U266, RPMI-8226, MM.1S, UM9) and the LUC⁺ Burkitt lymphoma cell line Raji, compared with isotype control (gray histogram). GPRC5D MFI is provided. (B) These cell lines were incubated with solvent control, serial dilutions of talquetamab (0.00128-4.0 µg/mL), or control antibodies, together with PB-MNCs obtained from healthy donors as effector cells, at an E:T ratio of 10:1 for 48 hours. Tumor cell lysis was assessed by using a BLI-based cytotoxicity assay. Data represent mean MM lysis ± standard error of mean (SEM) of 3 to 5 independent experiments, performed in duplicate. Half maximal effective concentration (EC₅₀) values are provided. (C) LUC⁺ RPMI-8226 cells were incubated for 48 hours with solvent control or serial dilutions of talquetamab (0.00128-4.0 µg/mL), and PB-MNCs obtained from healthy donors as effector cells at different E:T ratios (10:1, 3:1, 1:1, 1:3, and 1:10). MM cell lysis was determined by using a BLI-based cytotoxicity assay. Data represent mean MM lysis ± SEM of 3 independent experiments, performed in duplicate. (D) Talquetamab-mediated activation and degranulation of healthy CD4⁺ and CD8⁺ T cells were assessed by flow cytometric analysis of CD25 and CD107a cell-surface expression, respectively (n = 3). Representative flow cytometry histogram overlays depict cell surface expression of CD25 and CD107a on CD4⁺ (green) and CD8⁺ (purple) T cells treated with talquetamab (4.0 µg/mL) or solvent control (gray). (E) Pro-inflammatory cytokines and granzyme B were measured in cell-culture supernatants from RPMI-8226 cells treated with talquetamab (0.032-4.0 μg/mL) in the presence of PB-MNCs obtained from healthy donors for 48 hours, by using a flow cytometry-based assay and an enzyme-linked immunosorbent assay, respectively. Bars represent mean ± SEM of 3 independent experiments, performed in triplicate. (F) RPMI-8226 cells were incubated with solvent control or serial dilutions of talquetamab (0.00128-4.0 µg/mL), and PB-MNCs derived from 4 healthy donors or from 9 RRMM patients (median of 3 prior lines of therapy, 66% refractory for both IMiD and PI; PB was obtained at the time of progression during last line of therapy) at an E:T ratio of 10:1. After a 48 hour-incubation, lysis of RPMI-8826 cells was determined by BLI. Data represent mean MM lysis ± SEM; each individual experiment was performed in duplicate. Differences in talquetamab-mediated tumor cell lysis in the presence of PB-MNCs from healthy donors or MM patients were calculated using nonlinear regression analysis.

Figure 3.

Talquetamab-mediated lysis is hampered by the presence of BMSCs in 2 of 3 cell lines. (A) Luciferase-transduced cell lines RPMI-8226, MM.1S, and UM9 were incubated with solvent control, serial dilutions of talquetamab (0.00128-4.0 µg/mL), or control antibodies, and with PB-MNCs obtained from healthy donors at an E:T ratio of 10:1, in the absence or presence of BMSCs (ratio of BMSCs to MM cells was 2:1). After a 48-hour incubation, MM cell lysis was assessed using a BLI-based cytotoxicity assay. Data represent mean MM lysis ± SEM of 3 to 4 independent experiments performed in duplicate. Differences in talquetamab-mediated tumor cell lysis in the presence or absence of BMSCs were calculated using nonlinear regression analysis; P values are provided. (B) The indirect or direct impact of BMSCs on talquetamab-mediated lysis was evaluated by performing transwell experiments in which MM cells (RPMI-8226 or MM.1S cells) and PB-MNCs obtained from healthy donors were placed in the lower chambers, and no cells (medium alone) or BMSCs (indirect contact) were placed in the upper chambers. To study direct cell contact, all cell types were combined in the lower chambers. Cells were incubated with serial dilutions of talquetamab (0.032-4.0 µg/mL) or control antibodies for 48 hours. Lysis of MM cells was assessed using a BLI assay. Data represent mean lysis ± SEM of 3 independent experiments, performed in duplicate or triplicate. The impact of direct or indirect contact with BMSCs was calculated using nonlinear regression analysis and 2-way analysis of variance test; P values are provided. (C) GPR5D expression on MM cells was assessed by flow cytometry after a 48-hour incubation in the presence or absence of BMSCs. Paired Student t test was used to evaluate significance between both groups.

Figure 4.

Talquetamab-mediated lysis of primary MM cells. BM-MNCs obtained from 13 NDMM, 17 daratumumab-naïve RRMM, and 15 daratumumab-refractory RRMM patients were incubated with solvent control, serial dilutions of talquetamab (0.00128-4.0 µg/mL), and control antibodies (4.0 µg/mL) for 48 hours, after which surviving CD138⁺ MM cells (A), as well as T and NK cells (B), were enumerated using flow cytometric analysis. Lysis (y-axis) of MM cells (red), T cells (orange) and NK cells (gray) is depicted in the graphs. Negative lysis values indicate that cell numbers are higher when compared with solvent control. (C) Talquetamab-mediated tumor cell lysis in samples obtained from NDMM (lime green), daratumumab-naïve RRMM (light blue), and daratumumab-refractory RRMM (magenta) patients was compared using nonlinear regression analysis. EC₅₀ values are provided for each patient category. (D) Talquetamab-mediated activation and degranulation of CD4⁺ T cells, CD8⁺ T cells, and NK cells were assessed after a 48-hour culture in all 45 patient samples by flow cytometric analysis of CD25 and CD107a cell surface expression, respectively. Data represent mean ± SEM; experiments performed in duplicate. Representative flow cytometry histogram overlays depict cell surface expression of CD25 (green) and CD107a (purple) on CD4⁺ and CD8⁺ T cells treated with talquetamab (4.0 µg/mL) for 48 hours, compared with solvent control (gray). (E) T-cell activation and T-cell degranulation were also assessed per patient category (NDMM [lime green], daratumumab-naïve RRMM [light blue], and daratumumab-refractory RRMM [magenta]), and compared using nonlinear regression analysis. (F) Granzyme B and cytokines were measured in the cell-culture supernatants of BM-MNCs treated with solvent control or talquetamab for 48 hours. BM-MNCs were obtained from 5 daratumumab-naïve RRMM and 1 daratumumab-refractory RRMM patient. Data are depicted as different symbols, representing the mean of 4 measurements per patient, with box and whiskers, representing median values, 25th-75th percentile, and range. Data were normalized to solvent control. GZMB, granzyme B.

Figure 5.

Impact of tumor, patient and immune characteristics on talquetamab-mediated lysis, T-cell activation, and degranulation. The impact of tumor and patient characteristics (A) and immune characteristics (B) on talquetamab-mediated activity was assessed by constructing dose-response curves for talquetamab-mediated MM cell lysis (panels I), talquetamab-mediated T-cell activation based on frequency of CD25⁺ T cells (panels II), and talquetamab-mediated T-cell degranulation based on frequency of CD107a⁺ T cells (panels III), according to median expression levels (GPRC5D, BCMA), median percentage of immune cell subsets (T cells, Tregs, PD-1⁺ T cells, HLA-DR⁺ T cells, naïve T cells), median E:T ratio, median age, as well as presence of high-risk cytogenetic aberrations. Patient samples were obtained from 13 NDMM, 17 daratumumab-naïve RRMM, and 15 daratumumab-refractory RRMM patients. Data represent mean ± SEM, samples with a value ≤ median value of all tested samples are depicted in blue and > median value in red. Groups were compared by nonlinear regression analysis. P values are provided.

Figure 5.

Impact of tumor, patient and immune characteristics on talquetamab-mediated lysis, T-cell activation, and degranulation. The impact of tumor and patient characteristics (A) and immune characteristics (B) on talquetamab-mediated activity was assessed by constructing dose-response curves for talquetamab-mediated MM cell lysis (panels I), talquetamab-mediated T-cell activation based on frequency of CD25⁺ T cells (panels II), and talquetamab-mediated T-cell degranulation based on frequency of CD107a⁺ T cells (panels III), according to median expression levels (GPRC5D, BCMA), median percentage of immune cell subsets (T cells, Tregs, PD-1⁺ T cells, HLA-DR⁺ T cells, naïve T cells), median E:T ratio, median age, as well as presence of high-risk cytogenetic aberrations. Patient samples were obtained from 13 NDMM, 17 daratumumab-naïve RRMM, and 15 daratumumab-refractory RRMM patients. Data represent mean ± SEM, samples with a value ≤ median value of all tested samples are depicted in blue and > median value in red. Groups were compared by nonlinear regression analysis. P values are provided.

Figure 5.

Impact of tumor, patient and immune characteristics on talquetamab-mediated lysis, T-cell activation, and degranulation. The impact of tumor and patient characteristics (A) and immune characteristics (B) on talquetamab-mediated activity was assessed by constructing dose-response curves for talquetamab-mediated MM cell lysis (panels I), talquetamab-mediated T-cell activation based on frequency of CD25⁺ T cells (panels II), and talquetamab-mediated T-cell degranulation based on frequency of CD107a⁺ T cells (panels III), according to median expression levels (GPRC5D, BCMA), median percentage of immune cell subsets (T cells, Tregs, PD-1⁺ T cells, HLA-DR⁺ T cells, naïve T cells), median E:T ratio, median age, as well as presence of high-risk cytogenetic aberrations. Patient samples were obtained from 13 NDMM, 17 daratumumab-naïve RRMM, and 15 daratumumab-refractory RRMM patients. Data represent mean ± SEM, samples with a value ≤ median value of all tested samples are depicted in blue and > median value in red. Groups were compared by nonlinear regression analysis. P values are provided.

Figure 6.

Tregs suppress the proliferation and killing capacity of CD4⁺CD25^-effector T cells. (A) Tregs and CD4⁺CD25⁻ effector T cells were isolated from healthy donor-derived buffy coats by using an immune-magnetic cell isolation kit. Baseline immune cell frequencies and purity of isolated fractions were determined by flow cytometry, representative density plots are depicted. (B) Effector cells were labeled with violet tracer and incubated with or without Tregs in the presence of anti-CD3/CD28-coated beads. Proliferation was measured by flow cytometry after 5 days. (C) RPMI-8226 cells were incubated with Tregs and/or effector cells for 48 hours at an E:T ratio of 1:1 and 3:1, with solvent control or talquetamab (4.0 µg/mL). In combination experiments, Tregs and effector T cells were combined in a 1:1 ratio. Lysis of MM cells was determined using a BLI-based cytotoxicity assay. (D) Cytokine and granzyme B concentrations in cell-culture supernatants were measured using flow cytometry, and concentrations in talquetamab-treated conditions are depicted as fold change compared with solvent control. Data represent mean ± SEM of 3 independent experiments, performed in triplicate. Groups were compared using ANOVA with Tukey’s posttest. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 7.

Co-incubation with daratumumab (DARA) or pomalidomide (POM) enhances talquetamab-mediated lysis. (A) BM-MNCs obtained from 8 NDMM, 11 daratumumab-naïve RRMM. and 6 daratumumab-refractory RRMM patients were incubated for 48 hours with serial dilutions of talquetamab (0.00128-0.8 µg/mL) with or without daratumumab 0.1 µg/mL. Surviving MM cells were enumerated using flow cytometry. (B) The effect of talquetamab with or without daratumumab on the frequency of CD38⁺ T cells, CD38⁺ CD4⁺ T cells, and CD38⁺ CD8⁺ T cells was assessed by flow cytometric analysis. (C) The impact of talquetamab with or without daratumumab on T-cell lysis, T-cell activation (CD25 positivity), and T-cell degranulation (CD107a positivity) was determined by flow cytometry. (D) For pomalidomide experiments, BM-MNCs obtained from 5 NDMM, 1 RRMM, and 7 DARA-R MM patients were incubated for 48 hours with serial dilutions of talquetamab (0.00128-4.0 µg/mL) with or without pomalidomide (2 µM). MM cell lysis was assessed by flow cytometry. (E) T-cell lysis, T-cell activation (CD25 positivity) and T-cell degranulation (CD107a positivity) were measured by flow cytometry. (F) Granzyme B concentration was measured in the cell-culture supernatants of BM-MNCs, obtained from 5 patients, treated for 48 hours with serial dilutions of talquetamab (0.00128-0.8 µg/mL) with or without pomalidomide (2 µM). Granzyme B concentrations are depicted as fold change compared with solvent control. (B-C,E-F) Results of talquetamab monotherapy were compared with combinatorial treatment by nonlinear regression analysis with matched 2-way ANOVA and Sidak’s posttest. (A-F) Data are depicted as mean ± SEM of individual experiments, performed in duplicate. *P < .05; **P < .01; ***P < .001; ****P < .0001.