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

Abstract

BackgroundAberrant 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.MethodsAntibody 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.ResultsH-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.ConclusionsH-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.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0525-0) contains supplementary material, which is available to authorized users.

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

“…Results from the cynomolgus monkey confirm this finding, suggesting that anti-RON ADCs are highly stable in vivo [ 61 – 63 ]. The PK profiles of Zt/g4-MMAE in the cynomolgus monkey fit the two-compartment model [ 62 ]. Zt/g4-MMAE has an average mean plasma clearance of 0.12 ml/day/kg, a t½ of ~ 6.5 days, and a mean residential time of ~ 7.50 days in the cynomolgus monkey [ 62 ].…”

“…The PK profiles of Zt/g4-MMAE in the cynomolgus monkey fit the two-compartment model [ 62 ]. Zt/g4-MMAE has an average mean plasma clearance of 0.12 ml/day/kg, a t½ of ~ 6.5 days, and a mean residential time of ~ 7.50 days in the cynomolgus monkey [ 62 ]. These observations demonstrate that anti-RON ADCs are stable and display a favorable PK profile.…”

“…The maximal tolerance dose (MTD) for both MMAE- and DCM-based ADCs is ~ 60 mg/kg as judged by mouse daily activity, food consumption, and bodyweight. In monkeys, Zt/g4-MMAE at a single dose of 10 or 30 mg/kg does not cause clinical abnormalities judging from animal daily activity, bodyweight, body temperature, food consumption, heart rate, breath, vision, and urination [ 62 ]. Also, no evidence of tissue inflammation, cell death, structural alteration, hemorrhage, or other pathological changes has been observed in all animals tested [ 62 ].…”

“…In monkeys, Zt/g4-MMAE at a single dose of 10 or 30 mg/kg does not cause clinical abnormalities judging from animal daily activity, bodyweight, body temperature, food consumption, heart rate, breath, vision, and urination [ 62 ]. Also, no evidence of tissue inflammation, cell death, structural alteration, hemorrhage, or other pathological changes has been observed in all animals tested [ 62 ]. Nevertheless, adverse reactions from blood chemistry analysis indicate slight to moderate abnormalities in blood leukocytes, reticulocytes, and a panel of liver enzymatic activities [ 62 ].…”

“…Also, no evidence of tissue inflammation, cell death, structural alteration, hemorrhage, or other pathological changes has been observed in all animals tested [ 62 ]. Nevertheless, adverse reactions from blood chemistry analysis indicate slight to moderate abnormalities in blood leukocytes, reticulocytes, and a panel of liver enzymatic activities [ 62 ]. These effects are in a dose-dependent manner, reversible, and manageable.…”