Abstract
A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins—MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.
Highlights
A potential molecular target for aggressive cancers is the plasminogen activation system (PAS), which plays a key role in tissue degradation during cancer invasion
Flow cytometry of various cell lines indicated that MDA-MB-231 cells display the highest level of uPAR (Figure 1a), and have been previously shown to have higher uPAR RNA level expression compared to other breast cancer cell lines [18]
The binding of the human recombinant anti-uPAR 2G10 to uPAR was previously investigated [17], and we reported that 2G10 Fab and IgG binds uPAR using surface plasmon resonance with KD values of 10 × 10−9 and 2 × 10−12 mol/L, respectively [18]. 2G10 competes for binding with urokinase plasminogen activator (uPA), but it was not known whether the antibody binds uPAR in the uPA-binding site or in a distant site that induces a conformational change that prevents uPA binding. 2G10 does not recognize the reduced, unfolded uPAR as shown using Western blotting (See SDS-PAGE gels, Figure 1b)
Summary
A potential molecular target for aggressive cancers is the plasminogen activation system (PAS), which plays a key role in tissue degradation during cancer invasion. It has been known for over two decades that overexpression of the serine protease urokinase plasminogen activator (uPA) and its receptor uPAR contributes to the aggressive phenotype in a number of cancers such as breast (both invasive and non-invasive), lung, and gastrointestinal tumors and their metastases, and that this expression is not observed in non-diseased tissue [1,2,3,4,5,6]. Others have shown with both knockout studies and extensive work on mouse uPAR that significant toxicities are not seen when mouse uPAR function is blocked [12]
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