Preclinical validation of anti-TMEFF2-auristatin E–conjugated antibodies in the treatment of prostate cancer

Abstract

Current treatments for advanced stage, hormone-resistant prostate cancer are largely ineffective, leading to high patient mortality and morbidity. To fulfill this unmet medical need, we used global gene expression profiling to identify new potential antibody-drug conjugate (ADC) targets that showed maximal prostate cancer-specific expression. TMEFF2, a gene encoding a plasma membrane protein with two follistatin-like domains and one epidermal growth factor–like domain, had limited normal tissue distribution and was highly overexpressed in prostate cancer. Immunohistochemistry analysis using a specific monoclonal antibody (mAb) to human TMEFF2 showed significant protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease. To evaluate anti-TMEFF2 mAbs as potential ADCs, one mAb was conjugated to the cytotoxic agent auristatin E via a cathepsin B–sensitive valine-citrulline linker. This ADC, Pr1-vcMMAE, was used to treat male severe combined immunodeficient mice bearing xenografted LNCaP and CWR22 prostate cancers expressing TMEFF2. Doses of 3 to 10 mg/kg of this specific ADC resulted in significant and sustained tumor growth inhibition, whereas an isotype control ADC had no significant effect. Similar efficacy and specificity was shown with huPr1-vcMMAE, a humanized anti-TMEFF2 ADC. No overt in vivo toxicity was observed with either murine or human ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with the murine TMEFF2 protein, implying minimal toxicity to other body tissues. These data support the further evaluation and clinical testing of huPr1-vcMMAE as a novel therapeutic for the treatment of metastatic and hormone-resistant prostate cancer.

FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

“…However, we observed the colocalization of anti-TenB2− FRET3 conjugate with LysoTracker after the internalized vesicles were spread throughout the cytosol after 15 h (Figure S3b), which is in line with the previous observation that the TenB2 antigen eventually is localized in lysosomes in TenB2-PC3 cells. 27,32,36 In contrast, there was no directed clustering of internalized vesicles in SK-BR-3 cells (Figure 5a to 5c and Supporting Movie 1). We observed colocalization of the anti-HER2−FRET3 conjugates with LysoTracker, suggesting the conjugate reaches lysosomes (Figure S3a).…”

“…34,35 In contrast, the TenB2 antigen proceeded directly to the lysosomal pathway in PC3 cells with minimal recycling. 26,27,32 We hypothesized that our novel FRET-labeled ADCs may provide insight on whether these two different internalization pathways impact ADC processing.…”

“…34,35 In contrast, TenB2 antigens do not show as much recycling, and they proceed primarily to the lysosomes. 27,32,36 It still remains to be resolved whether or not the intracellular uptake pathway matters for the ADC potency. Recently, a specific DM1 transporter SLC46A3 has been identified in lysosomal membranes by Hemblett et al 46 It is possible that this DM1 transporter enhanced the cytosolic transport of the released DM1 payload specifically in SK-BR-3 cells.…”

“…PC3 cell lines have been engineered to express either the prostate cancer-specific growth factor receptor tomoregulin-2 or HER2 at the cell surface. 27 We compared the antigen-mediated internalization and processing of TenB2 and HER2 in the same PC3 cell background. In addition, we compared the processing of the same receptor, HER2, in the different cell backgrounds (SK-BR-3 and PC3 cells), to better understand drivers of the antigen-mediated antibody uptake pathway.…”

“…SK-BR-3 is a HER2-positive breast cancer cell line, while PC3 is a prostate cancer cell line. PC3 cell lines have been engineered to express either the prostate cancer-specific growth factor receptor tomoregulin-2 or HER2 at the cell surface . We compared the antigen-mediated internalization and processing of TenB2 and HER2 in the same PC3 cell background.…”