The Antitumor Activity of IMGN529, a CD37-Targeting Antibody-Drug Conjugate, Is Potentiated by Rituximab in Non-Hodgkin Lymphoma Models

Abstract

Naratuximab emtansine (IMGN529) is an investigational antibody-drug conjugate consisting of a CD37-targeting antibody conjugated to the maytansine-derived microtuble disruptor, DM1. IMGN529 has shown promising preclinical and clinical activity in non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Due to the aggressive nature of the disease, DLBCL is often treated with combination therapies to maximize clinical outcomes; therefore, we investigated the potential of combining IMGN529 with both standard-of-care and emerging therapies against multiple oncology-relevant targets and pathways. The strongest enhancement in potency was seen with anti-CD20 antibodies, including rituximab. The combination of IMGN529 and rituximab was more potent than either agent alone, and this combinatorial benefit was associated with increased apoptotic induction and cell death. Additional studies revealed that rituximab treatment increased the internalization and degradation of the CD37-targeting antibody moiety of IMGN529. The combination of IMGN529 and rituximab was highly efficacious in multiple xenograft models, with superior antitumor efficacy seen compared to either agent alone or treatment with R-CHOP therapy. These findings suggest a novel mechanism whereby the potency of IMGN529 can be enhanced by CD20 binding, which results in the increased internalization and degradation of IMGN529 leading to the generation of greater amounts of cytotoxic catabolite. Overall, these data provide a biological rationale for the enhanced activity of IMGN529 in combination with rituximab and support the ongoing clinical evaluation of IMGN529 in combination with rituximab in patients with relapsed and/or refractory DLBCL.

Figure 1

Synergy screening identifies combinatorial benefit between IMGN529 and anti-CD20 antibodies, which requires the intact ADC. (A) Heat map of synergy values for IMGN529 in combination with anti-CD20 antibodies. The cell line panel included ABC DLBCL (green), GCB DLBCL (orange), MCL (blue), and BL (gray) lines. (B) Synergy scores for rituximab in combination with the K7153A antibody, free DM1, or intact IMGN529 evaluated in U-2932, DOHH-2, and OCI-Ly18 cells.

Figure 2

Combination IMGN529 and anti-CD20 antibody treatment induces apoptosis in DLBCL cell lines. (A) Caspase 3/7 activation in DLBCL cell lines following overnight exposure to 2 nM rituximab or 0.125 nM IMGN529, alone and in combination. *P < .0001 (unpaired t test). (B) Western blot analysis of cleaved PARP expression in the same panel of lines treated with rituximab, IMGN529, or the combination for 24 hours; β-actin and GAPDH are included as loading controls. (C) Caspase 3/7 activation in U-2932 and SU-DHL-4 cells was determined following overnight exposure to anti-CD20 antibodies, K7153A, IgG1-SMCC-DM1, or IMGN529, alone and in combination as indicated.

Figure 3

ADCC, but not CDC, is augmented by the combination of IMGN529 and rituximab. ADCC assays were performed by incubating target U-2932 (A) or SU-DHL-4 (B) cells with human NK effector cells at E/T ratio of 3:1 or 4:1 and measuring LDH release. Cells were exposed to increasing concentrations of IMGN529 in the presence or absence of varying concentrations of rituximab or control IgG, as indicated. Percent specific lysis was calculated, and data shown are the mean of three experiments. (C) CDC activity against U-2932 cells (left panel) and SU-DHL-4 cells (right panel) was determined following incubating cells for 2 hours with increasing concentrations of rituximab +/− 5 μg/mL of IMGN529 or IgG1-SMCC-DM1 in the presence of human complement. Cell viability was assessed by alamarBlue assay.

Figure 4

Rituximab cotreatment increases the internalization of the CD37-targeting moiety of IMGN529. (A) U-2932 cells (left panel) and SU-DHL-4 cells (right panel) cells were incubated with AF488-labeled K7153A in the presence or absence of the indicated control or anti-CD20 antibodies at 37°C for 30 minutes, 2 hours, and 6 hours. The percent internalization was determined by flow cytometry (*P < .0001, unpaired t test). (B) The internalization of AF488-labeled rituximab was determined alone or in the presence of K7153A or anti-CD19 antibodies in U-2932 (left panel) and SU-DHL-4 cells (right panel) cells.

Figure 5

IMGN529 processing and catabolite formation are enhanced in the presence of rituximab. (A) U-2932 (left panel) and SU-DHL-4 cells (right panel) cells were pulse-treated with 10 nM ³H-K7153A either alone or in the presence of the indicated antibodies (10 nM) for 30 minutes and cultured for 24 hours at 37°C. Antibodies bound per cell values were calculated from resulting total sample CPM using the ³H- K7153A reagent-specific radioactivity, cell number, and assumption of 1:1 reagent per receptor binding. (B) U-2932 and SU-DHL-4 cells were treated with 10 nM of ³H-K7153A either alone or in the presence of the indicated antibodies (10 nM) for 30 minutes and cultured for 24 hours at 37°C. Cells and media were precipitated with acetone upon harvest, and the percentage processed was calculated from the CPM values of the supernatant (processed ³H-7153A) and pellet (intact ³H-7153A) (*P < .0001). (C) The amount of degraded ³H-Ab (catabolite) was calculated from the supernatant (processed ³H-Ab) CPM value and converted to picomoles of catabolite per million cells (*P < .0001).

Figure 6

Combination IMGN529 plus rituximab treatment confers superior antitumor efficacy in vivo in models of B-cell NHL. (A) SCID mice bearing U-2932 xenografts were dosed with vehicle, R-CHOP, a single dose of 10 mg/kg IMGN529, 10 mg/kg rituximab (QW×3), or the combination. (B) SCID mice bearing SU-DHL-4 xenografts were treated with a single dose of vehicle, R-CHOP, 10 mg/kg IMGN529, 10 mg/kg rituximab, or the combination, as indicated. (C) SCID mice bearing OCI-Ly18 xenografts were treated with a single dose of vehicle, 10 mg/kg IMGN529, 10 mg/kg rituximab, or the combination. (D) Kaplan-Meier analysis of overall survival in the Farage xenograft model. Beginning 7 days after tumor cell inoculation, SCID mice with disseminated lymphatic disease were dosed with vehicle, a single administration of 5 mg/kg IMGN529, 10 mg/kg rituximab (QW×3), or the combination, and animal survival was monitored out to 120 days.

Supplementary Figure 1.

Body weight changes in multiple DLBCL xenograft models. Mice bearing U-2932 (A), SU-DHL-4 (B), and OCI-Ly18 (C) xenograft tumors were treated as shown in Figure 6. Body weights were measured twice weekly.