Therapeutic efficacy of a novel humanized antibody-drug conjugate recognizing plexin-semaphorin-integrin domain in the RON receptor for targeted cancer therapy

Abstract

BackgroundAntibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application.MethodMonoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice.ResultsH5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC50 at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON+/CD44+/ESA+ phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg.ConclusionH5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.

MET and RON receptor tyrosine kinases in colorectal adenocarcinoma: molecular features as drug targets and antibody-drug conjugates for therapy

“…For ADCs to be effective, antibody-induced robust receptor internalization is essential to deliver sufficient payloads for cancer cell killing [ 55 , 56 ]. Third , targeted delivery of payloads by anti-MET and/or RON mAbs is highly effective in vitro in killing CRAC cells [ 57 – 63 ]. This effect is proportionally correlated with levels of MET and/or RON expression by CRAC cells.…”

“…In addition, the approach of using antibody-directed drug delivery overcomes the shortcomings that have occurred in SMKI- and TMAB-targeted CRAC therapy, which rely on addiction of MET and/or RON signaling for cellular survival [ 11 , 12 , 15 ]. Finally , studies from animal models have proven that anti-MET and anti-RON mAb-directed drug delivery inhibits CRAC xenograft growth, although the efficacy varies significantly [ 57 , 63 ].…”

“…RON-targeted ADCs such as Zt/g4-MMAE and PCM5B14-ducarmycin also have been preclinically validated (Fig. 3 ; Table 1 ) and are ready for clinical trials [ 61 – 63 , 79 – 81 ]. Moreover, a novel dual targeting ADC specific to both MET and RON has been validated for clinical development (Fig.…”

“… Antibodies selected for ADC development : Currently, two anti-RON mAbs, Zt/g4 and PCM5B14, have been selected, resulting in Zt/g4- and PCM5B14-based ADCs for potential clinical evaluation (Fig. 3 ) [ 61 – 63 , 79 – 81 ]. Both mAbs are specific to the RON extracellular domains.…”

“…Both mAbs induce a robust RON internalization by CRAC cells, which ensures sufficient amounts of payload delivered for cytotoxic activity and avoids potential development of chemoresistance. Both mAbs have been conjugated with different payloads such as DM1, MMAE, and DCM with proper conjugation profiles, serum stability, and PK parameters [ 61 – 63 , 79 – 81 ]. These features make Zt/g4 and PCM5B14 ideal candidates for anti-RON ADC development.…”