Abstract
Background: MORAb-202 is an antibody–drug conjugate (ADC) consisting of farletuzumab, a humanized anti-folate receptor-alpha (FRα) monoclonal antibody, conjugated to eribulin as a payload. MORAb-202 targets eribulin to tumor cells expressing FRα and is being investigated in clinical trials in patients with solid tumors. In the dose-escalation part of a first-in-human phase 1 study, partial response was observed beginning at MORAb-202 0.3 mg/kg. To evaluate the antitumor activity of MORAb-202, we investigated its biological mechanism, focusing on its bystander effect and ability to induce immunogenic cell death (ICD). Methods: The bystander effect of MORAb-202 was further characterized in an in vitro coculture system of the FRα− HL60 and FRα+ IGROV1 cell lines. Analyses included: 1) different ratios of FRα− cells from ∼0.5–2-fold against FRα+ cell line; 2) time-dependent bystander effect from 48 to 96 hours; 3) cell cytotoxicity of the FRα− cells in the presence of supernatant recovered from coculture condition; 4) bystander effect of anti-FRα−sulfo-SPDB-DM4 (FRα-DM4). In vitro induction of ICD by eribulin and by MORAb-202 was examined in breast cancer (BC) cell lines. In vivo ICD induction was examined in OV CDx and TNBC PDx models. Post-dose tumor samples from OV CDx and TNBC PDx models were analyzed by IHC/IF staining against HMGB1, HSP90, and F4/80 as ICD markers. Results: A consistent in vitro bystander effect of MORAb-202 was observed from 48 to 96 hours. Endpoint supernatant recovered from the coculture exhibited cell cytotoxicity in FRα− cells, suggesting that released eribulin is liable for the observed bystander effect. FRα-DM4 failed to induce a bystander effect; its off-target effect was greater than MORAb-202. Both eribulin and MORAb-202 elicited in vitro ICD induction of calreticulin and HMGB1 in human BC cell lines. In in vivo studies, MORAb-202 (12.5 mg/kg) treatment on OV CDx had durable tumor shrinkage; eribulin treatment (1.0 mg/kg) showed tumor regrowth. HSP90 staining from MORAb-202- and eribulin-treated tumors showed enhanced intensity from day 1 after treatment, while no HSP90 enhancement was observed in vehicle-treated tumor. HMGB1 staining from MORAb-202- and eribulin-treated tumors showed intensified cytoplasmic and membrane translocation from the nuclei, from day 1; no translocation of HMGB1 staining was observed in vehicle control. A lower dose of MORAb-202 (5 mg/kg) in TNBC PDx was examined, and enhanced staining and translocation of HMGB1 in all tumors treated with MORAb-202 was observed. Additionally, MORAb-202 treatment-accumulated macrophages (F4/80) were found in tumors. Conclusion: MORAb-202 exhibited a strong in vitro bystander effect with a limited off-target effect, while FRα-DM4 failed to show a bystander effect with a higher off-target effect. MORAb-202 induced ICD in both in vitro and in vivo settings. Conflict of interest: Other Substantive Relationships: Employees of Eisai Inc. - Brian Drozdowski, James Fulmer, Keiji Furuuchi, Katherine Rybinski, Allis Soto, and Toshimitsu Uenaka
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