The anti-tumoral potential of the saporin-based uPAR-targeting chimera ATF-SAP

Abstract

the development of personalized therapies represents an urgent need owing to the high rate of cancer recurrence and systemic toxicity of conventional drugs. So far, targeted toxins have shown promising results as potential therapeutic compounds. Specifically, toxins conjugated to antibodies or fused to growth factors/enzymes have been largely demonstrated to selectively address and kill cancer cells. We investigated the anti-tumor potential of a chimeric recombinant fusion protein formed by the Ribosome inactivating protein saporin (SAp) and the amino-terminal fragment (Atf) of the urokinasetype plasminogen activator (upA), whose receptor has been shown to be over-expressed on the surface of aggressive tumors. Atf-SAp was recombinantly produced by the P. pastoris yeast and its activity was assessed on a panel of bladder and breast cancer cell lines. Atf-SAp resulted to be highly active in vitro, as nano-molar concentrations were sufficient to impair viability on tumor cell lines. In contrast to untargeted toxins, the chimeric fusion protein displayed a significantly improved toxic effect in uPARexpressing cells, demonstrating that the selective activity was due to the presence of the targeting moiety. Fibroblasts were not sensitive to ATF-SAP despite uPAR expression, indicating that cell-specific receptor-mediated internalization pathway(s) might be considered. the in vivo anti-tumor effect of the chimera was shown in a bladder cancer xenograft model. Current findings indicate ATF-SAP as a suitable anti-tumoral therapeutic option to cope with cancer aggressiveness, as a single treatment or in combination with traditional therapeutic approaches, to appropriately address the intra-and intertumor heterogeneity.

Targeting the Urokinase-Type Plasminogen Activator Receptor (uPAR) in Human Diseases With a View to Non-invasive Imaging and Therapeutic Intervention

“…Initially, such intervention programs primarily focused on function inhibition to dampen surface associated plasminogen activation ( Crowley et al, 1993 ; Schmiedeberg et al, 2002 ; Lin et al, 2020 ; Yuan et al, 2021 ), but with the limited expression of uPAR in vital tissues, the focus gradually shifted toward targeted interventions based on cytotoxic eradication of uPAR expressing cells. Such uPAR-targeted treatment modalities include (i) recruiting the immune response to eliminate uPAR expressing cells using CAR-T cells ( Amor et al, 2020 ) or priming the adaptive immune response with uPAR-targeted haptens ( Rullo et al, 2016 ), (ii) proteolytic activation of prodrugs by uPAR-bound uPA ( Liu et al, 2003 ; Gerspach et al, 2006 ; Schafer et al, 2011 ), (iii) uPAR-mediated internalization of cytotoxin-conjugated uPA-derivatives ( Waldron et al, 2012 ; Zuppone et al, 2020 ) or antibodies ( Harel et al, 2019 ), and (iv) targeted radiotherapy ( Knör et al, 2008 ; Persson et al, 2012c ; LeBeau et al, 2013 ). Notwithstanding the low uPAR expression in most vital tissues, the baseline expression in the glomeruli of normal kidneys may, however, pose a concern for such cytotoxic treatment modalities.…”

“…In the last two decades, a variety of different targeted intervention strategies have been developed to eradicate uPAR-expressing cells. Specificity of the cytotoxic insult were devised by targeting uPAR directly or by exploiting the proteolytic activity of uPAR-bound uPA ( Liu et al, 2003 ; Rustamzadeh et al, 2007 ; Schafer et al, 2011 ; Morodomi et al, 2012 ; LeBeau et al, 2013 ; Jing et al, 2017 ; Harel et al, 2019 ; Amor et al, 2020 ; Zuppone et al, 2020 ). Several of these treatment modalities have shown promising results in preclinical mouse models bearing human cancer cell xenografts.…”

The Urokinase Receptor (uPAR) as a “Trojan Horse” in Targeted Cancer Therapy: Challenges and Opportunities

“…In vitro, selective cytotoxicity was demonstrated against uPAR-overexpressing U937 leukemia cells with a IC 50 of 0.1 nM [ 206 ]. Recently, the therapeutic potential of ATF-SAP has also been investigated in other putative candidate uPAR-overexpressing tumor entities, including breast and bladder cancers [ 207 ]. ATF-SAP selectively killed the in vitro models in a dose-dependent manner and proportionally to uPAR surface density, with IC 50 values in the nanomolar range, as found for the U937 cells [ 207 ].…”

Fibrinolytic System and Cancer: Diagnostic and Therapeutic Applications

“…In another study, a bispecific immunotoxin, DTATEGF, targeting the EGF/EGFR and uPA/uPAR axes showed a potent cytotoxic effect in human metastatic non-small cell lung cancer (NSCLC) brain tumor xenografts [119]. More recently, Zuppone et al showed that conjugation of a ribosome-inactivating protein called saporin (SAP) with ATF significantly reduced the viability of breast and bladder cancer cell lines [120]. Furthermore, the anti-cancer effects of the ATF-SAP conjugate were selective towards cancer cells with no discernable effect on the growth of non-tumorigenic fibroblast cells expressing high levels of uPAR.…”