Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

RON Receptor Tyrosine Kinase in Tumorigenic Stemness as a Therapeutic Target of Antibody-drug Conjugates for Eradication of Triple-negative Breast Cancer Stem Cells.

BACKGROUND: Cancer stem-like cells in triple-negative breast cancer (TNBC-SLCs) are the tumorigenic core for malignancy. Aberrant expression of the RON receptor tyrosine kinase has implications in TNBC tumorigenesis and malignancy. OBJECTIVE: In this study, we identified the RON receptor as a pathogenic factor contributing to TNBC cell stemness and validated anti-RON antibody-drug conjugate Zt/g4-MMAE for eradication of RONexpressing TNBC-SLCs. METHODS: Immunofluorescence and Western blotting were used for analyzing cellular marker expression. TNBC-SLCs were isolated by magnetic-immunofluorescence cell-sorting techniques. Spheroids were generated using the ultralow adhesion culture methods. Levels of TNBC-SLC chemosensitivity were determined by MTS assays. TNBC-SLC mediated tumor growth was determined in athymic nude mice. The effectiveness of Zt/g4-induced RON internalization was measured by immunofluorescence analysis. Efficacies of Zt/g4-MMAE in killing TNBC-SLCs in vitro and in eradicating TNBC-SLCmediated tumors were determined in mouse models. All data were statistically analyzed using the GraphPad Prism 7 software. RESULTS: Increased RON expression existed in TNBC-SLCs with CD44+/CD24- phenotypes and ALDH activities and facilitated epithelial to mesenchymal transition. RON-positive TNBC-SLCs enhanced spheroid-formatting capability compared to RON-negative TNBC-SLCs, which were sensitive to small molecule kinase inhibitor BMS-777607. Increased RON expression also promoted TNBC-SLC chemoresistance and facilitated tumor growth at an accelerated rate. In vitro, Zt/g4-MMAE caused massive TNBC-SLC death with an average IC50 value of ~1.56 µg per/ml and impaired TNBC cell spheroid formation. In mice, Zt/g4-MMAE effectively inhibited and/or eradicated TNBC-SLC mediated tumors in a single agent regimen. CONCLUSION: Sustained RON expression contributes to TNBC-SLC tumorigenesis. Zt/g4-MMAE is found to be effective in vivo in killing TNBC-SLC-mediated xenograft tumors. Our findings highlight the feasibility of Zt/g4-MMAE for the eradication of TNBC-SLCs in the future.

Antibody-drug Conjugate PCMC1D3-Duocarmycin SA as a Novel Therapeutic Entity for Targeted Treatment of Cancers Aberrantly Expressing MET Receptor Tyrosine Kinase.

BACKGROUND: Aberrant expression of the MET receptor tyrosine kinase is an oncogenic determinant and a drug target for cancer therapy. Currently, antibody-based biotherapeutics targeting MET are under clinical trials. OBJECTIVE: Here, we report the preclinical and therapeutic evaluation of a novel anti-MET antibody- drug conjugate PCMC1D3-duocarmycin SA (PCMC1D3-DCM) for targeted cancer therapy. METHODS: The monoclonal antibody PCMC1D3 (IgG1a/κ), generated by a hybridoma technique and specific to one of the MET extracellular domains, was selected based on its high specificity to human MET with a binding affinity of 1.60 nM. PCMC1D3 was conjugated to DCM via a cleavable valine-citrulline dipeptide linker to form an antibody-drug conjugate with a drug-to-antibody ratio of 3.6:1. PCMC1D3-DCM in vitro rapidly induced MET internalization with an internalization efficacy ranging from 6.5 to 17.2h dependent on individual cell lines. RESULTS: Studies using different types of cancer cell lines showed that PCMC1D3-DCM disrupted the cell cycle, reduced cell viability, and caused massive cell death within 96h after treatment initiation. The calculated IC50 values for cell viability reduction were 1.5 to 15.3 nM. Results from mouse xenograft tumor models demonstrated that PCMC1D3-DCM in a single dose injection at 10 mg/kg body weight effectively delayed xenograft tumor growth up to two weeks without signs of tumor regrowth. The calculated tumoristatic concentration, a minimal dose required to balance tumor growth and inhibition, was around 2 mg/kg body weight. Taken together, PCMC1D3-DCM was effective in targeting the inhibition of tumor growth in xenograft models. CONCLUSION: This work provides the basis for the development of humanized PCMC1D3-DCM for MET-targeted cancer therapy in the future.

Antibody-drug conjugates targeting RON receptor tyrosine kinase as a novel strategy for treatment of triple-negative breast cancer.

Treatment of triple-negative breast cancer (TNBC) is a challenge to oncologists. Currently, the lack of effective therapy has fostered a major effort to discover new targets and therapeutics to combat this disease. The recepteur d'origine nantais (RON) receptor has been implicated in the pathogenesis of TNBC. Clinical studies have revealed that aberrant RON expression is crucial in regulating TNBC malignant phenotypes. Increased RON expression also has prognostic value for breast cancer progress. These features provide the rationale to target RON for TNBC treatment. In this review, we discuss the importance of RON in TNBC tumorigenesis and the development of anti-RON antibody-drug conjugates (ADCs) for clinical application. The findings from preclinical studies lay the foundation for clinical trials of this novel biotherapeutic for TNBC therapy.

Targeting RON receptor tyrosine kinase for treatment of advanced solid cancers: antibody-drug conjugates as lead drug candidates for clinical trials.

The recepteur d'origine nantais (RON) receptor tyrosine kinase, belonging to the mesenchymal-to-epithelial transition proto-oncogene family, has been implicated in the pathogenesis of cancers derived from the colon, lung, breast, and pancreas. These findings lay the foundation for targeting RON for cancer treatment. However, development of RON-targeted therapeutics has not gained sufficient attention for the last decade. Although therapeutic monoclonal antibodies (TMABs) targeting RON have been validated in preclinical studies, results from clinical trials have met with limited success. This outcome diminishes pharmaceutical enthusiasm for further development of RON-targeted therapeutics. Recently, antibody-drug conjugates (ADCs) targeting RON have drawn special attention owing to their increased therapeutic activity. The rationale for developing anti-RON ADCs is based on the observation that cancer cells are not sufficiently addicted to RON signaling for survival. Thus, TMAB-mediated inhibition of RON signaling is ineffective for clinical application. In contrast, anti-RON ADCs combine a target-specific antibody with potent cytotoxins for cancer cell killing. This approach not only overcomes the shortcomings in TMAB-targeted therapies but also holds the promise for advancing anti-RON ADCs into clinical trials. In this review, we discuss the latest advancements in the development of anti-RON ADCs for targeted cancer therapy including drug conjugation profile, pharmacokinetic properties, cytotoxic effect in vitro, efficacy in tumor models, and toxicological activities in primates.

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

BACKGROUND: Antibody-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. METHOD: Monoclonal 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. RESULTS: H5B14 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. CONCLUSION: H5B14-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.

Correction to: Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy.

Following publication of the original article [1], the author reported two errors in the authors affiliations.

Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy.

BACKGROUND: Aberrant expression of the RON receptor tyrosine kinase is a pathogenic feature and a validated drug target in various types of cancers. Currently, therapeutic antibodies targeting RON for cancer therapy are under intensive evaluation. Here we report the development and validation of a novel humanized anti-RON antibody-drug conjugate for cancer therapy. METHODS: Antibody humanization was achieved by grafting sequences of complementarity-determining regions from mouse monoclonal antibody Zt/g4 into human IgG1/κ acceptor frameworks. The selected humanized Zt/g4 subclone H1L3 was conjugated with monomethyl auristatin E using a dipeptide linker to form H-Zt/g4-MMAE. Pharmacokinetic analysis of H-Zt/g4-MMAE was determined using hydrophobic interaction chromatography and a MMAE ADC ELISA kit. Biochemical and biological assays were used for measuring RON expression, internalization, cell viability and death. Therapeutic efficacies of H-Zt/g4-MMAE were validated in vivo using three pancreatic cancer xenograft models. Toxicological activities of H-Zt/g4-MMAE were determined in mouse and cynomolgus monkey. RESULTS: H-Zt/g4-MMAE had a drug to antibody ratio of 3.77:1 and was highly stable in human plasma with a dissociation rate less than 5% within a 20 day period. H-Zt/g4-MMAE displayed a favorable pharmacokinetic profile in both mouse and cynomolgus monkey. In vitro, H-Zt/g4-MMAE induced RON internalization, which results in killing of pancreatic cancer cells with IC50 values at 10-20 nM. In vivo, H-Zt/g4-MMAE inhibited pancreatic cancer xenograft growth with tumoristatic concentrations at 1~3 mg/kg bodyweight. Significantly, H-Zt/g4-MMAE eradicated tumors across multiple xenograft models regardless their chemoresistant and metastatic statuses. Moreover, H-Zt/g4-MMAE inhibited and eradicated xenografts mediated by pancreatic cancer stem-like cells and by primary cells from patient-derived tumors. Toxicologically, H-Zt/g4-MMAE is well tolerated in mice up to 60 mg/kg. In cynomolgus monkey, H-Zt/g4-MMAE up to 30 mg/kg had a manageable and reversible toxicity profile. CONCLUSIONS: H-Zt/g4-MMAE is superior in eradication of pancreatic cancer xenografts with favorable pharmacokinetic profiles and manageable toxicological activities. These findings warrant the transition of H-Zt/g4-MMAE into clinical trials in the future.

RON Receptor Tyrosine Kinase as a Therapeutic Target for Eradication of Triple-Negative Breast Cancer: Efficacy of Anti-RON ADC Zt/g4-MMAE.

Triple-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. We analyzed RON expression in 168 primary TNBC samples via tissue microarray using anti-RON IHC staining and demonstrated that RON was widely expressed in 76.8% TNBC samples with overexpression in 76 cases (45.2%). These results provide the molecular basis to target RON for TNBC therapy. To this end, anti-RON monoclonal antibody Zt/g4-drug monomethyl auristatin E conjugate (Zt/g4-MMAE) was developed with a drug to antibody ratio of 3.29 and tested in a panel of TNBC cell lines with different phenotypes. 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/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. In vivo, Zt/g4-MMAE at 10 mg/kg in a Q12 × 2 regimen completely eradicated TNBC xenografts without the regrowth of xenograft tumors. In conclusion, increased RON expression is a pathogenic feature in primary TNBC samples. Zt/g4-MMAE is highly effective in eradicating TNBC xenografts in preclinical models. These findings lay the foundation for using anti-RON Zt/g4-MMAE in clinical trials as a novel strategy for TNBC treatment.

Preclinical Efficacy of Anti-RON Antibody-Drug Conjugate Zt/g4-MMAE for Targeted Therapy of Pancreatic Cancer Overexpressing RON Receptor Tyrosine Kinase.

Aberrant expression of the RON receptor tyrosine kinase, a cell surface protein, is a pathogenic feature in pancreatic cancer, which renders it a drug target for targeted therapy. Nevertheless, development of therapeutics targeting RON for pancreatic cancer therapy is hampered due to the lack of full addiction by pancreatic cancer cells to RON signaling for growth and survival. Here we describe a novel strategy using anti-RON antibody-directed drug delivery in the form of an antibody-drug conjugate for inhibition and/or eradication of pancreatic cancers. Monoclonal antibody Zt/g4 specific to the RON Sema domain was selected as the drug carrier based on its ability to induce robust RON internalization. Conjugation of Zt/g4 with monomethyl auristatin E, designated as Zt/g4-MMAE, was achieved through a protease-sensitive dipeptide linker to reach a drug to antibody ratio of 3.29:1. Zt/g4-MMAE was stable in human plasma with a dissociation rate less than 4% within a 10 day period. In vitro, Zt/g4-MMAE rapidly induced RON internalization, resulting in cell cycle arrest followed by massive cell death. The maximal effect was seen in pancreatic cancer cells with more than 10 000 receptor molecules per cell. Zt/g4-MMAE also synergized in vitro with chemotherapeutics including gemcitabine, 5-fluorouracil, and oxaliplatin to further reduce PDAC cell viability. In vivo, Zt/g4-MMAE exerts a long-lasting activity, which not only inhibited but also eradicated pancreatic xenograft tumors. These finding indicate that Zt/g4-directed drug delivery is highly effective for eradicating pancreatic tumors. Thus, Zt/g4-MMAE is a novel biotherapeutic with potential for therapy of RON-expressing pancreatic malignancies.