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

Background The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin 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 Amphiregulin. 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, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin 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 Amphiregulin. 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 Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. Conclusions This ADC targeting Amphiregulin has potential utility in the treatment of breast and other tumors in which proteolytic Amphiregulin shedding is a frequent event.

Cross-species meta-analysis of transcriptome changes during the morula to blastocyst transition: metabolic and physiological changes take center stage

The morula to blastocyst transition (MBT) culminates with formation of inner cell mass (ICM) and trophectoderm (TE) lineages. Recent studies identified signaling pathways driving lineage specification, but some features of these pathways display significant species divergence. To better understand evolutionary conservation of the MBT, we completed a meta-analysis of RNA sequencing data from five model species and ICM-TE differences from four species. While many genes change in expression during the MBT within any given species, the number of shared DEGs is comparatively small, and the number of shared ICM-TE DEGs is even smaller. DEGs related to known lineage determining pathways (e.g., POU5F1) are seen, but the most prominent pathways and functions associated with shared DEGs or shared across individual species DEG lists impact basic physiological and metabolic activities, such as TCA cycle, unfolded protein response, oxidative phosphorylation, sirtuin signaling, mitotic roles of polo-like kinases, NRF2-mediated oxidative stress, estrogen receptor signaling, apoptosis, necrosis, lipid and fatty acid metabolism, cholesterol biosynthesis, endocytosis, AMPK signaling, homeostasis, transcription, and cell death. We also observed prominent differences in transcriptome regulation between ungulates and non-ungulates, particularly for ICM- and TE-enhanced mRNAs. These results extend our understanding of shared mechanisms of the MBT and ICM/TE formation and should better inform the selection of model species for particular applications.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-β signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified β-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by β-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm’s canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17β-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17β-estradiol in AH supports a role for estrogen signaling in IOP regulation.

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

The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin 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. Here, we report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin 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 Amphiregulin. Antibodies conjugated with monomethyl auristatin E (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 Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection.