Transcatheter arterial chemoembolization (TACE) has become the preferred treatment for unresectable advanced hepatocellular carcinoma (HCC). However, embolization of the tumor-supplying artery during TACE often leads to hypoxia-associated tumor angiogenesis, which limits the therapeutic outcome for HCC. In this study, Wangdeng et al. [1] utilized a vascular endothelial growth factor receptor (VEGFR)-targeting peptide, VEGF125-136 (QKRKRKKSRYKS), combined with a lytic peptide (KLUKLUKKLUKLUKLUK) to form a peptide-drug conjugate (PDC). The study employed cellular affinity assays to assess the binding capacity of this peptide to VEGFR-overexpressing cell lines, and used CCK8 and apoptosis assays to confirm cytotoxicity across different cell lines. A VX2 tumor-bearing rabbit model was established to evaluate the in vivo anti-tumor efficacy of the peptide conjugate in combination with TACE. The peptide conjugate not only targeted cell surface VEGFR and inhibited VEGFR function but also exhibited potent anti-cancer effects. The study found that this peptide conjugate demonstrated strong cytotoxicity against HCC cells (Huh7; IC50 of 7.3 ± 0.74 μM) and endothelial cells (HUVEC; IC50 of 10.7 ± 0.292 μM), and induced apoptosis in both cell lines. Furthermore, in the VX2 rabbit tumor model, these peptides administered via TACE showed superior in vivo anti-tumor efficacy compared to the conventional drug doxorubicin (DOX), effectively inhibited angiogenesis in tumor tissue, and exhibited a favorable safety profile. This research may provide an alternative strategy for clinical HCC therapy through TACE combination therapy.

Design and Mechanism of Action of the Peptide-Drug Conjugate (PDC)

Anti-angiogenesis in tumor tissue has become a key strategy for TACE. In addition to small molecule drugs, many peptide ligands exhibit good binding affinity for VEGFR and have been widely used for anti-angiogenic therapy in tumors or as radiotracers for malignancies. This study selected a reported potent VEGFR peptide inhibitor, VEGF125-136 (QKRKRKKSRYKS, designated QR), to inhibit the interaction between VEGF and VEGFR. However, the inhibitory effect of this peptide alone was insufficient to suppress tumor proliferation. Therefore, a lytic peptide (KLUKLUKKLUKLUK, designated KLU) was selected and conjugated to this VEGFR peptide ligand, termed QR-KLU, as shown in Figure 1. This designed peptide could not only inhibit VEGFR signaling but also suppress tumor proliferation through non-specific membrane disruption. This designed peptide conjugate could be combined with TAE for the treatment of HCC, representing a novel and potent therapeutic agent.

Figure 1: Schematic diagram of the QR-KLU peptide conjugate

Affinity of the QR-KLU Peptide for Endothelial Cells

Cellular competitive binding assays were performed to evaluate the affinity of the peptide conjugate for endothelial cells (HUVEC) with high VEGFR expression. FAM-labeled VEGF125-136 peptide QR was co-incubated with HUVEC cells for 4 hours, and cell penetration ability was then assessed by flow cytometry. As shown in Figure 2A, at 0 μM QR-KLU, the FAM-labeled QR peptide demonstrated strong cell penetration; however, cell penetration ability decreased with increasing concentrations of QR-KLU. These results indicate that at low concentrations, QR-KLU can compete with the VEGF peptide for binding to VEGFR on the cell surface, suggesting strong binding affinity of the QR-KLU peptide for endothelial cells. To further confirm these results, the experiment was repeated using HT29 cells, which have low VEGFR expression. It was found that cell penetration ability of the FAM-labeled QR peptide was lower in HT29 cells compared to HUVEC cells (Figures 2B, C). Furthermore, different concentrations of QR-KLU peptide did not significantly affect the cell penetration ability of the FAM-labeled QR peptide. Collectively, these data indicate that the QR-KLU peptide has stronger affinity for endothelial cells that highly express VEGFR.

Figure 2: Cellular affinity of QR-KLU for different cell lines

Cytotoxicity of the QR-KLU Peptide

The effect of the QR-KLU peptide conjugate on the proliferation of Huh7 and HUVEC cells was evaluated using the CCK8 assay. First, the anti-proliferative capacity of three peptides (QR, KLU, and QR-KLU) against Huh7 and HUVEC cells was investigated at various concentrations. As shown in Figures 3A and 3B, cells were treated with QR peptide, KLU peptide, QR-KLU peptide, or DOX at different concentrations. In both cell lines, the inhibition rate in the KLU and QR-KLU groups increased in a dose-dependent manner. In HUVEC cells, QR-KLU (IC50 of 10.7 ± 0.292 μM) exhibited stronger inhibitory activity than KLU (IC50 of 33.8 ± 0.98 μM). In Huh7 cells, QR-KLU (IC50 of 7.3 ± 0.74 μM) also exhibited stronger anti-tumor activity than KLU (IC50 of 36.27 ± 2.7 μM). Meanwhile, the QR peptide showed negligible toxicity at concentrations up to 80 μM. As expected, DOX exhibited the strongest cytotoxicity in both cell lines (IC50 of 0.243 ± 0.076 μM for Huh7 cells; IC50 of 2.12 ± 0.72 μM for HUVEC cells). Collectively, these data indicate that QR-KLU possesses significant cytotoxicity in vitro. These results demonstrate that the designed QR-KLU peptide conjugate can significantly inhibit the proliferation of both cancer cells and endothelial cells.

Figure 3: Cytotoxicity validation of the QR-KLU peptide

Effect of QR-KLU on Apoptosis of Huh7 and HUVEC Cells

The enhanced pro-apoptotic activity of QR-KLU was further confirmed by Annexin V-PI staining using flow cytometry. Live cells, early apoptotic cells, necrotic cells, and late apoptotic cells are represented as Q4, Q3, Q2, and Q1, respectively. At a concentration of 10 μM, the QR-KLU peptide exhibited a significant pro-apoptotic effect, with apoptosis rates exceeding 60% in Huh7 cells and exceeding 40% in HUVEC cells (Figure 4). Furthermore, QR-KLU induced apoptosis in a dose-dependent manner. However, at a concentration of 80 μM, the QR peptide did not show a significant pro-apoptotic effect in either Huh7 or HUVEC cells. At a KLU peptide concentration of 20 μM, the percentage of apoptotic cells was approximately 25% in both Huh7 and HUVEC cells.

Figure 4: Validation of the effect of QR-KLU on apoptosis of Huh7 and HUVEC cells

Tumor Growth 

A VX2 rabbit tumor model was established for TAE treatment. The average tumor volumes in the NS, DOX, KLU, and QR-KLU groups were 930.7 ± 461.1, 889.0 ± 317.7, 991.8 ± 114.2, and 899.7 ± 394.0 mm3, respectively (Figure 5A). Seven days after TAE treatment, the average tumor volumes in the four groups were 2540.5 ± 1173.8, 1382.9 ± 563.7, 1321.4 ± 210.4, and 1008.5 ± 370.7 mm3, respectively. Tumor volume in the NS group increased significantly, with an average growth rate of 276.4%, while tumor growth rates in the DOX, KLU, and QR-KLU groups were significantly lower than that in the NS group (P < 0.001), as shown in Figure 5B. Notably, the average tumor growth rate in the QR-KLU group was significantly lower than that in the other three groups (P < 0.01), while there was no significant difference between the KLU and DOX groups (P > 0.05).

Figure 5: Validation of QR-KLU inhibition of tumor growth

This study provides a new option for TACE treatment of HCC. Compared to conventional chemotherapeutic agents, the QR-KLU peptide exhibits stronger anti-tumor activity and anti-angiogenic effects, with a favorable safety profile. Therefore, the QR-KLU peptide holds significant potential for application in TACE therapy for liver cancer, warranting further consideration and investigation.

Tek Biotech (Tianjin) Co., Ltd. has established a comprehensive targeted peptide library construction and screening service platform based on phage display technology. This includes library construction and screening services for peptides of various lengths, such as linear and cyclic peptides, utilizing diverse screening methods (including solid-phase screening, magnetic bead screening, and in vivo animal screening). Additionally, we provide a full range of supporting downstream validation services, including affinity validation, blocking validation, and in vivo animal imaging validation, offering robust support for our clients' targeted small molecule discovery projects.

References

[1] Wang, D., Liu, J., Li, T. et al. A VEGFR targeting peptide-drug conjugate (PDC) suppresses tumor angiogenesis in a TACE model for hepatocellular carcinoma therapy. Cell Death Discov. 8, 411 (2022).