RON Receptor‐Targeted Antibody‐Drug Conjugate Therapy Ablates Cancer Stem Cells and Induces Long‐term Tumor Regressions in Preclinical Models of Triple‐Negative Breast Cancer (TNBC)

Abstract

PurposeTriple‐negative breast cancer (TNBC) is a highly diverse group of malignant neoplasia with poor outcome. Currently, the lack of effective therapy has fostered a major effort to discover new targets to treat this malignant cancer. Here we identified the RON receptor tyrosine kinase as a therapeutic target for potential TNBC treatment by using a novel anti‐RON monoclonal antibody Zt/g4‐drug monomethyl auristatin E conjugate (anti‐RON ADC Zt/g4‐MMAE)MethodsAnti‐RON antibody Zt/g4 was conjugated through a dipeptide linker to MMAE, a potent tubulin inhibitor. The generated Zt/g4‐MMAE has a drug‐antibody ratio of 3.2:1. The conjugation profile and drug stability were analyzed using HIC methods. A panel of TNBC cell lines representing different subtypes and expressing different levels of RON was used as a biological model. Various in vitro and in vivo experiments were performed to determine Zt/g4‐MMAE‐induced cell surface RON internalization, cell‐cycle arrest, and cellular cytotoxicity. The efficacy of Zt/g4‐MMAE was evaluated in TNBC xenograft models.ResultsWe first analyzed RON expression in 168 primary TNBC samples via tissue microarray using anti‐RON immunohistochemical staining and demonstrated that RON was widely expressed in 76.8 % TNBC samples with overexpression in 76 cases (45.2%). These findings provide the molecular basis to target RON for TNBC therapy. Anti‐RON monoclonal antibody Zt/g4‐drug monomethyl auristatin E conjugate (Zt/g4‐MMAE) was developed and tested in a panel of TNBC cell lines. In vitro, Zt/g4‐MMAE rapidly induced RON internalization, resulted in cell cycle arrest followed by massive cell death. The calculated IC50 values ranged from 0.06 to 3.46 μg per ml dependent on individual TNBC cell lines tested. Zt/g4‐MMAE also effectively killed TNBC stem‐like cells with RON+/CD44+/CD24− phenotypes and RON‐negative TNBC cells through the bystander effect, which effectively eliminated CSC subpopulation and reduced sphere formation of TNBC stem like‐cells. In vivo, Zt/g4‐MMAE at 10 mg/kg in a Q12 x 2 regimen completely eradicated TNBC xenografts caused by two TNBC cell lines without the regrowth of xenograft tumors.ConclusionIdentification of RON as the target opens a new avenue for TNBC treatment. Increased RON expression is a pathogenic feature in primary TNBC samples. Zt/g4‐MMAE is highly effective in eradicating TNBC xenografts in preclinical models with long lasting effect up to 6 weeks without signs of tumor regrowth. The current study has met our objectives. The first was to determine levels of RON expression from primary TNBC samples by anti‐RON immunohistochemical (IHC) staining. The second was to validate the efficacy of anti‐RON antibody Zt/g4‐drug monomethyl auristatin E (MMAE) conjugate for TNBC therapy. These studies provide the basis for targeting RON as a novel strategy for TNBC treatment using anti‐RON ADC Zt/g4‐MMAE.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.