A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma

Abstract

T-cell-mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein-coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).

Conflict of interest statement

Conflict-of-interest disclosure: All authors are employees of Janssen Pharmaceuticals and have ownership interest in Johnson & Johnson.

© 2020 by The American Society of Hematology.

Figures

Professional illustration by Katherine St. John.

Figure 1.

GPRC5D is selectively expressed on the surface of MM cells. (A-B) A bioinformatic analysis of 2 NCI public data sets shows upregulation of GPRC5D mRNA expression on MM patient CD138+ cells as well as SMM and plasma-cell leukemia samples (PCL). mRNA expression was plotted on log2 scale on the y-axis. (C) GPRC5D cell-surface protein expression on MM cells (AMO-1, EJM, H929, MM.1R, MOLP-8, and OPM2), B cells (Daudi), and myeloid cells (KG-1 and MOLM-13) was labeled using an anti-GPRC5D antibody (red line; 571961 clone; R&D Systems) or an isotype control antibody (gray line; IC0041A clone; R&D Systems) for 30 minutes at 4°C and analyzed by flow cytometry. (D) Images of cells with representative distribution of GPRC5D staining on the cell surface are shown. Merged images of GPRC5D staining and bright field are used to evaluate the localization of protein expression on the cell surface. (E) GPRC5D receptor density quantitation of CD138+ BM MNCs from healthy (n = 21) and MM patient samples (n = 23) shows averages of 1600 to 1800 receptors/cell (QuantiBRITE kit; difference is not statistically significant [N.S.]). (F) GPRC5D mRNA levels in various human tissues relative to H929 cells. Samples were analyzed for GPRC5D and glyceraldehyde 3-phosphate dehydrogenase mRNA expression using a TaqMan probe, and relative levels were plotted on the y-axis. Elevated levels of GPRC5D mRNA expression were observed in lymph node and spleen with minimal levels in lung, skin, and testis. The majority of the tissues were negative for GPRC5D. MGUS, monoclonal gammopathy of undetermined significance. GSE, Genomic Spatial Event database.

Figure 2.

JNJ-64407564 specifically kills GPRC5D+ target cells with concomitant T-cell activation. (A) JNJ-64407564 mediated cytotoxicity of GPRC5D+ cells (MM.1R, OPM-2, and H929) but not GPRC5D− cells (NALM-6 and Daudi), when incubated with healthy purified T cells at a 5:1 E/T ratio. Cytotoxicity was calculated as the percent of remaining CFSC-labeled cells compared with PBS controls after 48-hour incubation with the DuoBody antibodies. JNJ-64407564 (black circles) efficiently killed GPRC5D+ cells, but not GPRC5D− cells, while control antibodies GPRC5DxNull (red triangle) and NullxCD3 (blue square) had no effect. (B) 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. JNJ-64407564 activated T cells efficiently when incubated with GPRC5D+ cells, but not GPRC5D− cells, while control antibodies GPRC5DxNull and NullxCD3 had no effect. (C) Supernatants from the H929 assays were evaluated for cytokine levels using the Meso Scale Discovery human proinflammatory panel 1 kit (K15049D; Meso Scale Discovery). Average EC50 values from 6 different donor T cells are listed in the table.

Figure 3.

Expression of GPRC5D and BCMA on MM cells. (A) MM patient BM MNCs showing various profiles of GPRC5D and BCMA expression. Cells were gated using the CD138 plasma-cell marker (MI15; BioLegend) and antibodies to GPRC5D and BCMA to measure their surface expression (GPRC5D: 571961, R&D Systems; BCMA: 19F2, BioLegend). The left panel is representative of most patient samples surveyed, while the middle and right panels depict skewed expression for BCMA or GPRC5D, which was seen in a minority of patients (see panel B). (B) Expression of GPRC5D and BCMA surface protein in 51 BM MNC samples from MM patients on CD138+ cells as measured by flow cytometry. Each column represents an individual patient sample. Data are expressed as percent positivity of all CD138+ cells. (C) Generation of GPRC5D (top row) and BCMA (bottom row) KO H929 cells by CRISPR. Loss of signal can be seen in the KO cells compared with the isotype control (gray). (D) JNJ-64407564 (black circle) efficiently depleted H929 wild-type or BCMA KO cells, but not GPRC5D KO cells. Percent cytotoxicity and T-cell activation were measured as in Figure 2. A positive control BCMAxCD3 antibody (red triangle) killed wild-type H929 and GPRC5D KO cells but had no effect on BCMA KO cells. The negative control antibody NullxCD3 (blue square) had no effect on cytotoxicity or T-cell activation in all cell lines.

Figure 3.

Expression of GPRC5D and BCMA on MM cells. (A) MM patient BM MNCs showing various profiles of GPRC5D and BCMA expression. Cells were gated using the CD138 plasma-cell marker (MI15; BioLegend) and antibodies to GPRC5D and BCMA to measure their surface expression (GPRC5D: 571961, R&D Systems; BCMA: 19F2, BioLegend). The left panel is representative of most patient samples surveyed, while the middle and right panels depict skewed expression for BCMA or GPRC5D, which was seen in a minority of patients (see panel B). (B) Expression of GPRC5D and BCMA surface protein in 51 BM MNC samples from MM patients on CD138+ cells as measured by flow cytometry. Each column represents an individual patient sample. Data are expressed as percent positivity of all CD138+ cells. (C) Generation of GPRC5D (top row) and BCMA (bottom row) KO H929 cells by CRISPR. Loss of signal can be seen in the KO cells compared with the isotype control (gray). (D) JNJ-64407564 (black circle) efficiently depleted H929 wild-type or BCMA KO cells, but not GPRC5D KO cells. Percent cytotoxicity and T-cell activation were measured as in Figure 2. A positive control BCMAxCD3 antibody (red triangle) killed wild-type H929 and GPRC5D KO cells but had no effect on BCMA KO cells. The negative control antibody NullxCD3 (blue square) had no effect on cytotoxicity or T-cell activation in all cell lines.

Figure 4.

JNJ-64407564 can deplete MM cells when incubated with healthy human whole blood. (A-B) Human MM cells (H929) were incubated for 48 hours with whole blood from 6 different healthy donors at a 5:1 E/T ratio in the presence of various concentrations of JNJ-64407564. Percent cytotoxicity and T-cell activation were calculated as in Figure 2. (C) Healthy whole blood was incubated with JNJ-64407564 (black circle), GPRC5DxNull (white square), and NullxCD3 (black triangle) for 48 hours without exogenous target cells. (D) Binding of JNJ-64407564 to different cell populations in healthy human whole blood. The antibody was incubated with whole blood at 4°C for 30 minutes, and the binding profile was measured by flow cytometry (JNJ-64407564, red trace; IgG4-PAA isotype control, gray filled). Binding was captured using a PE-labeled secondary antibody (HP6025; SouthernBiotech).

Figure 5.

JNJ-64407564 can bind and deplete primary MM patient BM CD138+ cells. Frozen BM MNCs were incubated with various concentrations of JNJ-64407564 (0-532 nM) with or without exogenous healthy T cells to measure target binding and killing. Dose-dependent binding of JNJ-64407564 (black circles) and GPRC5DxNull (blue square) to target cells (top). NullxCD3 (red triangle) failed to show any binding. Dose-dependent plasma-cell depletion (middle). BM MNCs were incubated for 48 hours with exogenous healthy T cells at a 1:1 E/T ratio in the presence of JNJ-64407564 and depletion measured as remaining CD138+ and BCMA+ cells. JNJ-64407564-mediated T-cell activation was 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. JNJ-64407564 (circles) was able to activate T cells efficiently when incubated with GPRC5D+ cells, but not GPRC5D− cells, while control antibodies GPRC5DxNull (triangle) and NullxCD3 (square) had no effect.

Figure 6.

JNJ-64407564 can inhibit and regress tumors in murine models of MM. (A) JNJ-64407564 inhibits H929 tumor growth in a prophylactic xenograft model. NSG mice were injected IV with 1 × 107 human PBMCs, and after 7 days, each mouse received SC injection of 5 × 106 H929 cells in the right flank followed by IV administration of PBS (black circles) or JNJ-64407564 antibody at 0.1 µg (0.005 mg/kg, green downward triangle), 1 µg (0.05 mg/kg, red upward triangle), or 10 µg (0.5 mg/kg, blue square) per animal on days 0, 3, 5, 7, and 10. By day 19, the mean tumor volume of the PBS-treated control group (n = 6) had exceeded 600 mm3, and the mice were terminated. (B) JNJ-64407564 causes tumor regression in MM.1S MM xenograft model. Each mouse received 1 × 107 MM.1S cells in PBS in a total volume of 0.2 mL. Cells were implanted SC in the right flank using a 1-cm3 syringe and a 26-gauge needle. The day of tumor cell implantation was designated as day 0. On day 7 after tumor cell implant, animals were randomized with a tumor volume of ∼75 mm3 and received IV injection of 1 × 107 human PBMCs. Treatments were initiated on day 15, with each mouse receiving IV administration of PBS (black square) or JNJ-64407564 at 0.1 µg (0.005 mg/kg, blue circle), 1 µg (0.05 mg/kg, green upward triangle), 10 µg (0.5 mg/kg, red downward triangle), or 50 µg (2.5 mg/kg, orange diamond). Null bispecific antibody controls, GPRC5DxNull (dark green open square) and NullxCD3 (purple open circle), were each dosed at 10 µg per mouse. Treatments were administered for a total of 7 doses on days 15, 18, 22, 24, 29, 32, and 36 (indicated as “x” on the x-axis). Tumor measurements were taken up to day 36, when 80% to 100% of mice remained on study for each group. SEM, standard error of the mean.

Figure 7.

GPRC5D+ cells are depleted from MM.1S tumors. (A-B) A repeat MM.1S study was conducted testing 1 dose (10 μg) of JNJ-64407564 and control bispecific antibodies. (A) Tumor and blood samples were analyzed on the day after dosing on days 16 and 19 for tumor-cell number by staining with anti-BCMA antibody (percent remaining human CD45+ and BCMA+ cells on the y-axis; first column) and T-cell activation (percent CD25+ and CD4+ or CD8+ T cells on the y-axis; remaining columns). The BCMA antibody was used as a second plasma-cell marker to avoid using a GPRC5D antibody in the ex vivo testing. (B) T-cell (CD3+, CD4+, CD8+; SP57, Ventana) infiltration within the tumors. For each section, the left panel is low magnification (1.5×) and the right panel is a higher magnification (10×).