Transarterial chemoembolization (TACE) is a standard treatment for unresectable, advanced Hepatocellular Carcinoma (HCC), the most common type of liver cancer. However, a major limitation of TACE is that the embolization of tumor-feeding arteries can induce hypoxia, triggering tumor angiogenesis (new blood vessel growth) and ultimately limiting the treatment's overall effectiveness. Addressing this challenge, a recent study by Wang et al. (2022) developed a innovative Peptide-Drug Conjugate (PDC) designed to target this process. This new PDC combines a peptide that targets the Vascular Endothelial Growth Factor Receptor (VEGFR), a key driver of angiogenesis, with a cell-lytic peptide. The research demonstrates this conjugate's potent anti-tumor and anti-angiogenic effects, both in cell cultures and in a live animal model, suggesting a promising new avenue for combination therapy with TACE in HCC treatment.

Peptide-Drug Conjugate (PDC) Design and Mechanism

Inhibiting blood vessel formation within tumors is a crucial strategy to improve TACE outcomes. While small-molecule drugs are common, certain peptide ligands also show high binding affinity for VEGFR. The researchers selected a known potent VEGFR peptide inhibitor, VEGF125?136 (sequence: QKRKRKKSRYHS, named QR), to block the interaction between VEGF and its receptor. However, this inhibition alone was insufficient to halt tumor proliferation effectively.

To enhance efficacy, the team conjugated the QR peptide with a potent lytic peptide (sequence: KLUKLUKKLUKLUKLUK, named KLU), creating the PDC named QR-KLU. This dual-action design allows the conjugate not only to inhibit the VEGFR signaling pathway but also to directly kill cancer cells through non-specific membrane disruption. When used in conjunction with TACE, this PDC represents a novel and potent therapeutic approach for HCC.

*Figure 1: Schematic diagram of the QR-KLU peptide-drug conjugate design.

The study first assessed the PDC's ability to bind to endothelial cells, which highly express VEGFR. In a competitive binding assay, a FAM-labeled QR peptide was incubated with Human Umbilical Vein Endothelial Cells (HUVECs) in the presence of increasing concentrations of unlabeled QR-KLU. Flow cytometry analysis revealed that as the concentration of QR-KLU increased, the cell penetration of the FAM-labeled QR peptide decreased (Figure 2A). This indicates that QR-KLU successfully competes for binding sites on the VEGFR, demonstrating its high binding affinity for these cells.

To confirm target specificity, the experiment was repeated using HT29 cells, a line with low VEGFR expression. In these cells, the FAM-labeled QR peptide showed inherently lower penetration, which was unaffected by the presence of QR-KLU (Figure 2B, C). These results collectively confirm that the QR-KLU peptide has a strong and specific affinity for cells that highly express VEGFR.

*Figure 2: Cell binding affinity of QR-KLU in different cell lines (HUVEC vs. HT29).*

Cytotoxicity of the QR-KLU Peptide

The anti-proliferative effects of QR-KLU on both liver cancer cells (Huh7) and endothelial cells (HUVEC) were evaluated using the CCK-8 assay. The researchers tested QR, KLU, and the conjugated QR-KLU at various concentrations.

The results showed that both KLU and QR-KLU inhibited cell proliferation in a dose-dependent manner in both cell lines (Figure 3A, B). However, QR-KLU was significantly more potent than the KLU peptide alone. The IC50 values (concentration causing 50% inhibition) for QR-KLU were 7.3 ± 0.74 μM in Huh7 cells and 10.7 ± 0.292 μM in HUVEC cells, compared to 36.27 ± 2.7 μM and 33.8 ± 0.98 μM for KLU alone, respectively. The QR peptide alone showed negligible toxicity even at high concentrations (80 μM). As expected, the conventional chemotherapy drug Doxorubicin (DOX) showed the strongest cytotoxicity. These data confirm that the conjugated QR-KLU peptide has significant cytotoxic effects on both cancer and endothelial cells in vitro.

QR-KLU Induces Apoptosis in Huh7 and HUVEC Cells

The pro-apoptotic (cell death-inducing) activity of QR-KLU was further confirmed using Annexin V-PI staining and flow cytometry. At a concentration of 10 μM, QR-KLU induced significant apoptosis in over 60% of Huh7 cells and over 40% of HUVEC cells (Figure 4). This apoptotic effect was dose-dependent. In contrast, the QR peptide at a high concentration (80 μM) showed no significant pro-apoptotic effect, and the KLU peptide induced apoptosis in approximately 25% of cells at 20 μM. This demonstrates that the conjugated QR-KLU is highly effective at triggering programmed cell death.

In Vivo Tumor Growth Inhibition

The efficacy of QR-KLU was evaluated in vivo using a VX2 rabbit liver tumor model subjected to TACE-like procedures (TAE). Rabbits were treated with Normal Saline (NS), DOX, KLU, or QR-KLU.

Initial average tumor volumes were similar across all groups. Seven days after TAE treatment, the NS group showed a dramatic increase in tumor volume (average growth rate of 276.4%). In contrast, the DOX, KLU, and QR-KLU groups all showed significantly lower tumor growth rates (Figure 5A, B). Most importantly, the QR-KLU group demonstrated a significantly lower average tumor growth rate than all other groups, including DOX and KLU alone. There was no significant difference between the DOX and KLU groups. This indicates that QR-KLU is superior to both the standard drug and the lytic peptide alone in suppressing tumor growth in a live model.

Conclusion and Future Perspectives

This groundbreaking research provides a promising new option for enhancing TACE therapy in Hepatocellular Carcinoma. The novel Peptide-Drug Conjugate QR-KLU demonstrates superior anti-tumor and anti-angiogenic activity compared to traditional chemotherapy, with a favorable safety profile. By specifically targeting VEGFR and directly lysing tumor cells, QR-KLU addresses key limitations of conventional TACE. This PDC approach holds significant potential for clinical application in HCC treatment and warrants further investigation and development.

About TekBiotech

TekBiotech(Tianjin) Co., Ltd. leverages advanced phage display technology to offer comprehensive services for targeted peptide library construction and screening. Our platform includes the development and screening of diverse peptide libraries (including linear and cyclic peptides of varying lengths) using various methods such as solid-phase screening, magnetic bead screening, and in vivo animal screening. We also provide a suite of downstream validation services, including affinity validation, blocking assays, and in vivo animal imaging, to provide robust support for our clients' targeted drug discovery projects.

Reference

[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). https://doi.org/10.1038/s41420-022-01199-8