Correction: Yagolovich et al. DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma. Int. J. Mol. Sci. 2022, 23, 12687.

In the original publication [...].

Assessment of the effects of amphiphilic poly (N­vinylpyrrolidone) nanoparticles loaded with bortezomib on glioblastoma cell lines and zebrafish embryos.

Proteasome inhibitor bortezomib is an anticancer agent approved for treatment of multiple myeloma and mantle cell lymphoma. However, its application in other types of cancer, primarily in solid tumors, is limited due to poor pharmacokinetics, inefficient tissue penetration, low stability and frequent adverse effects. In the present study, a novel micellar nano-scaled delivery system was manufactured, composed of amphiphilic poly(N-vinylpyrrolidone) nanoparticles loaded with bortezomib. Similar nanoparticles loaded with prothionamide, a drug without anticancer effect, were used as control. The size and zeta potential of the obtained polymeric micelles were measured by dynamic light scattering. Bortezomib-loaded micelles exhibited significant cytotoxic activity in vitro in monolayer tumor cell cultures (IC50 ~6.5 µg/ml) and in 3D multicellular tumor spheroids (IC50 ~8.5 µg/ml) of human glioblastoma cell lines U87 and T98G. Additionally, the toxic effects in vivo were studied in zebrafish Danio rerio embryos, with an estimated 50% lethal concentration of 0.1 mg/ml. Considering that bortezomib and other molecules from the class of proteasome inhibitors are potent antitumor agents, nanodelivery approach can help reduce adverse effects and expand the range of its applications for treatment of various oncological diseases.

Dual targeting of DR5 and VEGFR2 molecular pathways by multivalent fusion protein significantly suppresses tumor growth and angiogenesis.

Destroying tumor vasculature is a relevant therapeutic strategy due to its involvement in tumor progression. However, adaptive resistance to approved antiangiogenic drugs targeting VEGF/VEGFR pathway requires the recruitment of additional targets. In this aspect, targeting TRAIL pathway is promising as it is an important component of the immune system involved in tumor immunosurveillance. For dual targeting of malignant cells and tumor vascular microenvironment, we designed a multivalent fusion protein SRH-DR5-B-iRGD with antiangiogenic VEGFR2-specific peptide SRH at the N-terminus and a tumor-targeting and -penetrating peptide iRGD at the C-terminus of receptor-selective TRAIL variant DR5-B. SRH-DR5-B-iRGD obtained high affinity for DR5, VEGFR2 and αvß3 integrin in nanomolar range. Fusion of DR5-B with effector peptides accelerated DR5 receptor internalization rate upon ligand binding. Antitumor efficacy was evaluated in vitro in human tumor cell lines and primary patient-derived glioblastoma neurospheres, and in vivo in xenograft mouse model of human glioblastoma. Multivalent binding of SRH-DR5-B-iRGD fusion efficiently stimulated DR5-mediated tumor cell death via caspase-dependent mechanism, suppressed xenograft tumor growth by >80 %, doubled the lifespan of xenograft animals, and inhibited tumor vascularization. Therefore, targeting DR5 and VEGFR2 molecular pathways with SRH-DR5-B-iRGD protein may provide a novel therapeutic approach for treatment of solid tumors.

DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma.

TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvß3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma.

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH-DR5-B with Dual VEGFR2 and DR5 Receptor Specificity.

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH-DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH-DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH-DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH-DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH-DR5-B can be considered as a promising candidate for therapeutic applications.