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Anti-tumor efficacy of an MMAE-conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer.

ABSTRACT:

Background

The Epidermal Growth Factor Receptor (EGFR) ligand, Amphiregulin (AREG), is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. AREG is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized.

Methods

Using phage display, we identified antibodies that selectively recognize the residual transmembrane stalk of cleaved AREG. Conjugation with fluorescence labels and monomethyl auristatin E (MMAE) was used to study their intracellular trafficking and anti-cancer effects, respectively.

Results

We report the development of an antibody-drug conjugate (ADC), GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved AREG, providing a novel means of targeting cells with high rates of AREG shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved AREG. Antibodies conjugated with MMAE were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the AREG neo-epitope in formalin-fixed, paraffin-embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection.

Conclusions

This ADC targeting AREG has potential utility in the treatment of breast and other tumors in which proteolytic AREG shedding is a frequent event.

SUBMITTER: Lofgren KA

PROVIDER: S-EPMC8643873 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.