A Novel RGD-4C-Saporin Conjugate Inhibits Tumor Growth in Mouse Models of Bladder Cancer

Abstract

Although toxin may have some advantages compared to chemotherapeutic drugs in cancer therapy, e.g. a potent cytotoxic activity and a reduced risk of resistance, their successful application in the treatments to solid tumors still remains to be fully demonstrated. In this study, we genetically modified the structure of the plant-derived single-chain ribosome inactivating protein saporin (SAP) by fusing its N-terminus to the ACDCRGDCFCG peptide (RGD-4C), an αv-integrin ligand, and explored the anti-tumor activity of the resulting protein (called RGD-SAP) in vitro and in vivo, using a model of muscle invasive bladder cancer. We found that the RGD-4C targeting domain enhances the cytotoxic activity of SAP against various tumor cell lines, in a manner dependent on αv-integrin expression levels. In a subcutaneous syngeneic model of bladder cancer, RGD-SAP significantly reduced tumor growth in a dose-dependent manner. Furthermore, systemic administration of RGD-SAP in combination with mitomycin C, a chemotherapeutic drug currently used to treat patients with bladder cancer, increased the survival of mice bearing orthotopic bladder cancer with no evidence of systemic toxicity. Overall, the results suggest that RGD-SAP represents an efficient drug that could be exploited, either alone or in combination with the state-of-the-art therapies, for the treatment of bladder cancer and, potentially, of other solid tumors.

RGD peptide in cancer targeting: Benefits, challenges, solutions, and possible integrin–RGD interactions

“…[137][138][139] A combination of RGD peptide-based therapies with other treatments like chemotherapy agents or immune checkpoint inhibitors could enhance treatment efficacy while potentially mitigating resistance mechanisms associated with single-agent therapy. 26,[140][141][142] Identifying biomarkers indicative of high integrin expression levels corresponding to likely responders allows for better patient stratification and personalized treatment approaches. Determining the optimal dosing, frequency, and routes of administration would maximize therapeutic efficacy while minimizing off-target effects on normal tissues with lower integrin expression levels.…”

“…Determining the optimal dosing, frequency, and routes of administration would maximize therapeutic efficacy while minimizing off-target effects on normal tissues with lower integrin expression levels. [142][143][144][145] Utilizing imaging techniques like PET or MRI in conjunction with RGD-based probes for visualizing changes in integrin expression during therapy allows early identification of responders and facilitates timely adjustments to the treatment plan if necessary. 53,[146][147][148] By employing these strategies individually or in combination, it is possible to overcome challenges posed by heterogeneous integrin expression when using | 9 of 44 RGD peptides for targeted therapies while maximizing their clinical potential.…”

“…resulting from receptor‐ligand interaction can release an active RGD molecule at targeted sites 137–139 . A combination of RGD peptide‐based therapies with other treatments like chemotherapy agents or immune checkpoint inhibitors could enhance treatment efficacy while potentially mitigating resistance mechanisms associated with single‐agent therapy 26,140–142 …”

“…Identifying biomarkers indicative of high integrin expression levels corresponding to likely responders allows for better patient stratification and personalized treatment approaches. Determining the optimal dosing, frequency, and routes of administration would maximize therapeutic efficacy while minimizing off‐target effects on normal tissues with lower integrin expression levels 142–145 . Utilizing imaging techniques like PET or MRI in conjunction with RGD‐based probes for visualizing changes in integrin expression during therapy allows early identification of responders and facilitates timely adjustments to the treatment plan if necessary 53,146–148 .…”

Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity

“…To achieve this goal, we used a 25 kDa plant derived globular protein, saporin (SAP), whose enzymatic activity is directed at inhibiting protein synthesis. [26][27][28][29] First, we used a fluorescently labelled protein, SAP-FITC, and an nsEV-to-cargo ratio of 1:1.5 (w:w). Strikingly, while in the case of electroporation, SAP-FITC incorporation was 4-5 times improved compared to that of dextran, reaching a loading efficiency of 1.5%, sodium carbonate treatment reached a 50 folds higher loading capacity of SAP-FITC with respect to dextran, significantly overperforming the electroporation (Figure 3C) and reaching a loading efficiency of 15%.…”