Ramantamig (JNJ-79635322), a novel T-cell-engaging trispecific antibody targeting BCMA, GPRC5D, and CD3, in multiple myeloma models

Abstract

In multiple myeloma (MM), cell-specific antigens are valuable targets for developing effective T-cell-engaging therapeutics that can provide good immune responses. Achieving a sustained immune response in recurrent MM, however, remains challenging. Ramantamig (JNJ-79635322) is a trispecific antibody targeting BCMA (B-cell maturation antigen) and GPRC5D (G-protein-coupled receptor family C group 5 member D), both of which are highly expressed on plasmablasts and plasma cells in samples from patients with myeloma. Dual antigen recognition on malignant plasma cells by a trispecific T-cell-engaging antibody could potentially enhance tumor binding through increased avidity, resulting in efficient depletion of the malignant clonal populations, targeting of tumor heterogeneity, and prevention of tumor antigen loss-mediated resistance. At subnanomolar ranges, ramantamig induced potent cytotoxicity in cancer cell lines with concomitant T-cell activation. Ramantamig efficiently depleted both dual- and single-target-expressing MM cell lines. In addition, it induced dose-dependent depletion of malignant plasma cells in samples from patients with MM both in an ex vivo T-cell coculture assay and in healthy fresh whole blood cocultured with H929 MM cells to mimic physiological conditions. Ramantamig exhibited potent antitumor activity in a murine xenograft prevention model (single-target-expressing clonal cells) and 2 tumor regression models. The potent and selective antitumor activity of ramantamig, with a clonal-depleting ability in vitro, ex vivo, and in vivo, warrants clinical evaluation of its ability to induce durable responses in myeloma. Phase 1 clinical trials are ongoing for patients with relapsed/refractory MM. These trials are registered at www.clinicaltrials.gov as NCT05652335 and NCT06768489.

Graphical abstract

Figure 1.

Ramantamig (JNJ-79635322)–mediated cell cytotoxicity and T-cell activation in target positive and target negative cell lines. The TsAb ramantamig (JNJ-79635322) (A) mediated cell cytotoxicity (B) and T-cell activation (C) in BCMA-GPRC5D dual-positive and BCMA-GPRC5D dual-negative cell lines. Panel A shows the structure of ramantamig (JNJ79635322). In panel B, ramantamig mediated cytotoxicity of dual-target–positive (BCMA and GPRC5D) cells (MM.1S, H929, JIM-3, and OPM-2) but not dual-target–negative (BCMA and GPRC5D) cells (MV-4-11 and OCI-AML-3) when incubated with healthy purified T cells at an effector-to-target (E:T) ratio of 3:1. Cytotoxicity was calculated as the percentage of remaining carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled cells compared with PBS controls after 72 hours of incubation with the TsAb. Ramantamig (JNJ-79635322; circles, orange trace) efficiently depleted dual-target–positive cells but not target-negative cells, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had no effect. In panel C, T-cell activation as measured by flow cytometry is shown. T cells were gated using the CD3 surface marker and CD25 activation marker. Percent CD25⁺ T-cell values were plotted on the y-axis. Ramantamig (JNJ-79635322) activated T cells efficiently when incubated with dual-target–positive cells but not with target-negative cells, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had no effect. The data points closely aligned with the generated fit curve with minimal donor-to-donor (n = 6 donors) variability.

Figure 2.

Ramantamig (JNJ-79635322) effects on T-cell activation in the absence of target cells. T-cell activation in T-cell (A) and whole blood samples (B), and ramantamig (JNJ-79635322) binding to hematological cells in whole blood (C). In panel A, ramantamig (JNJ-79635322)–mediated nonspecific T-cell activation was measured by incubating the TsAb mixed with purified T cells from 6 different donors for 48 hours and measured by flow cytometry. T cells were gated using the CD3 surface marker and CD25 activation marker. Percent CD25⁺ T-cell values were plotted on the y-axis. Neither ramantamig (JNJ-79635322; circles, orange trace) nor control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had any effect, as expected, due to the lack of myeloma target engagement. In panel B, a similar experiment was performed using whole blood from 6 different healthy donors to evaluate ramantamig (JNJ-79635322)–mediated nonspecific T-cell activation. Ramantamig (JNJ-79635322; circles, orange trace) did not have any effect on T-cell activation, as expected, due to the lack of myeloma target (BCMA or GPRC5D) engagement. In panel C, to evaluate the nonspecific cytotoxicity profile of ramantamig (JNJ-79635322), it was incubated with the whole blood from 6 different healthy donors for 48 hours, and percent live cells were analyzed through flow cytometry. The panel of 8 graphs depicts viability of immune cell subsets. The percent live (percent of all cells) cells was defined as the frequency of live cells in each immune cell subset in relation to the entire cell population, after red blood cell lysis of whole blood. Ramantamig (JNJ-79635322; circles, light blue trace) did not have any effect on cytotoxicity, as expected, due to the lack of myeloma target (BCMA or GPRC5D) engagement. NK, natural killer; NKT, natural killer T cells.

Figure 3.

Ramantamig (JNJ-79635322)–mediated cell cytotoxicity and T-cell activation in H929-WT, H929-GPRC5D-KO, and H929-BCMA-KO cell lines. Cytotoxicity mediated by ramantamig (JNJ-79635322; circles, orange trace) efficiently depleted H929-WT, H929-BCMA-KO, or H929-GPRC5D-KO cells (top). Concomitant T-cell activation was observed (bottom). No lysis or T-cell activation was observed with the control antibodies CD3×Null×Null (upward triangles, blue trace) or BCMA×GPRC5D×Null (downward triangles, red trace). Cells were incubated at E:T ratio of 3:1 for 72 hours at 37°C in a CO₂ incubator. The data points closely aligned with the generated fit curve with minimal donor-to-donor (n = 4 donors) variability. The effects of teclistamab (diamonds, purple trace) and talquetamab (hexagons, green trace) were also tested as positive controls.

Figure 4.

Ramantamig (JNJ-79635322)–mediated cell cytotoxicity and T-cell activation at different E:T ratios and time points. Cytotoxicity of ramantamig (JNJ-79635322) using H929 cells at various E:T ratios (A) and incubation times (B). In panel A, ramantamig (JNJ-79635322)–mediated cytotoxicity of dual-target–positive (BCMA and GPRC5D) H929 cells at various E:T ratios when incubated with healthy purified T cells for 72 hours. Cytotoxicity was calculated as the percent and listed on the y-axis. Ramantamig (JNJ-79635322; circles, orange trace) efficiently lysed dual-target–positive H929 cells, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had no effect. T-cell activation as measured by flow cytometry. T cells were gated using the CD3 surface marker and CD25 activation marker. Percent CD25⁺ T-cell values were plotted on the y-axis. Ramantamig (JNJ-79635322) (circles, orange trace) activated T cells efficiently, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had no effect. In panel B, cytotoxicity and T-cell activation measured after coincubating target H929 cells and T cells at 2 different E:T ratios of 3:1 and 1:1 in the presence of ramantamig (JNJ-79635322). The experiment involved incubation at various time points (24 hours, gray trace; 48 hours, red trace; 72 hours, dark blue trace; 96 hours, magenta trace; 120 hours, green trace, 144 hours, brown trace; and 168 hours, light blue trace), and cytotoxicity and T-cell activation was measured as described in the E:T ratio experiment shown in panel A.

Figure 5.

Cytotoxicity and T-cell activation potential of ramantamig (JNJ-79635322) against dual-target–positive H929 cells spiked in healthy human whole blood. Human MM cells (H929) were incubated for 48 hours with whole blood from 7 different healthy donors at an E:T ratio of 5:1 in the presence of various concentrations of ramantamig (JNJ-79635322). Cytotoxicity was calculated as the percent of remaining CFSE-labeled cells compared with PBS controls after 48 hours of incubation with the TsAb. Ramantamig (JNJ-79635322; circles, orange trace) efficiently lysed dual-target–positive H929 cells, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had no effect (top left graph). T-cell activation in T-cell subsets (CD3, CD4, and CD8) as measured by flow cytometry (top right graph and bottom panel). T cells were gated using the CD3, CD4, and CD8 surface markers and the CD25 activation marker. Percent CD25⁺ T-cell values were plotted on the y-axis. Ramantamig (JNJ-79635322) (circles, orange trace) activated T cells efficiently, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) and BCMA×GPRC5D×Null (downward triangles, red trace) had no effect. The data points closely aligned with the generated fit curve with minimal donor-to-donor (n = 6 donors) variability.

Figure 6.

Cytotoxicity of ramantamig (JNJ-79635322) against human primary CD138⁺ cells derived from patients with MM. Frozen BM MNC were incubated with various concentrations of ramantamig (JNJ-79635322; 0nM-532nM) with exogenous healthy T cells at an E:T ratio of 1:1 for 48 hours, and depletion was measured as remaining CD138⁺ cells. Dose-dependent plasma cell depletion, with percent plasma cell depletion listed on the y-axis (top). Ramantamig (JNJ-79635322)–mediated T-cell activation as measured by flow cytometry by gating T cells using CD3 surface marker and CD25 activation marker (bottom). Percent CD25⁺ T-cell values were plotted on the y-axis. Ramantamig (JNJ-79635322; circles, orange trace) was able to deplete plasma cells and activate T cells efficiently, whereas control antibodies CD3×Null×Null (upward triangles, blue trace) had no or minimal effect. MM BM MNC samples and healthy donor T cells were purchased from commercial vendors and coincubated for the cytotoxicity assay. BM, bone marrow; MNC, mononuclear cells.

Figure 7.

Ramantamig (JNJ-79635322)–mediated tumor regression and inhibition in xenograft models. Antitumor efficacy of ramantamig (JNJ-79635322) in subcutaneous (SC) RPMI 8226 xenografts (A), established SC MM1.S xenografts (B), and SC H929-BCMA-KO and H929-GPRC5D-KO xenografts (C) in T-cell–humanized NSG mice. In panel A, T-cell–humanized NSG mice bearing established SC RPMI 8226 tumors were IP dosed with PBS (light blue squares) and JNJ-79635322 at 0.1 (orange diamonds), 0.4 (green upward triangles), 1 (red downward triangles), and 2.5 mg/kg (dark blue circles). Treatments were administered on days 19, 21, 25, 28, 31, 34, 38, and 41 (indicated by the black arrows beneath the x-axis). Tumor volume was measured twice weekly, and the results were presented as the mean tumor volume ± standard error of the mean (SEM) for each group. Data were graphically represented for each group with at least two-thirds of the animals remaining in the study. In panel B, T-cell–humanized NSG mice bearing established SC MM.1S tumors were IP dosed with PBS (light blue squares) and ramantamig (JNJ-79635322) at 0.025 (orange circles), 0.1 (green upward triangles), 0.5 (red circles), and 1 mg/kg (dark blue upward triangles). Treatments were administered on days 15, 19, 22, 26, 30, 34, and 37 (indicated by the black line beneath the x-axis). Tumor volume was measured twice weekly, and the results were presented as the mean tumor volume ± SEM for each group. Data were graphically represented for each group with at least two-thirds of the animals remaining in the study. In panel C, T-cell–humanized NSG mice bearing SC H929-BCMA-KO (left flank) and SC H929-GPRC5D-KO (right flank) tumors were administered IP doses with PBS (light blue squares) and JNJ-79635322 at 0.025 (orange circles), 0.1 (green triangles), 0.25 (red circles), and 0.5 mg/kg (dark blue triangles). Treatments were administered on days 1, 5, 9, 13, 16, 19, and 22 (indicated by the black line beneath the x-axis). Tumor volume was measured twice weekly, and the results were presented as the mean tumor volume ± SEM for each group. Data were graphically represented for each group with at least two-thirds of the animals remaining in the study.