Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice.

CD44v6, a splice variant of the cell surface glycoprotein CD44, acts as a co-receptor for c-Met and is upregulated in tumors with high metastatic potential. Methods: We screened a phage-displayed peptide library for peptides that selectively bind to CD44v6-overexpressing cells and exploited them to block CD44v6 and deliver a pro-apoptotic peptide to tumors for cancer therapy. Results: CNLNTIDTC (NLN) and CNEWQLKSC (NEW) peptides bound preferentially to CD44v6-high cells than to CD44v6-low cells. The binding affinities of NLN and NEW to CD44v6 protein were 253 ± 79 and 85 ± 18 nM, respectively. Peptide binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments. Conclusions: These results suggest that NLN and NEW are promising metastasis-inhibiting peptide therapeutics and targeting moieties for CD44v6-expressing metastases.

Inhibition of Tumor Growth against Chemoresistant Cholangiocarcinoma by a Proapoptotic Peptide Targeting Interleukin-4 Receptor.

Cholangiocarcinoma (CCA) has a poor prognosis and high chemoresistance. Interleukin-4 receptor (IL-4R) is overexpressed in several cancer cells and plays a crucial role in tumor progression and drug resistance. IL4RPep-1, an IL-4R-binding peptide, has been identified by phage display and used for tumor targeting. In this study, we exploited IL4RPep-1 to guide the tumor-specific delivery of a proapoptotic peptide to chemoresistant CCA, thereby inhibiting tumor growth. Immunohistochemistry of human primary CCA tissues showed that IL-4R levels were upregulated in moderately to poorly differentiated types, and higher levels of IL-4R are correlated with lower survival rates in patients with CCA. IL4RPep-1 was observed to preferentially bind with high IL-4R-expressing KKU-213 human CCA cells, whereas it barely bound with low IL-4R-expressing KKU-055 cells. A hybrid of IL4RPep-1 and a proapoptotic peptide (KLAKLAK)2 (named as IL4RPep-1-KLA) induced cytotoxicity and apoptosis in KKU-213 cells and increased those levels induced by 5-fluorouracil (5-FU). IL4RPep-1-KLA was internalized in the cells and colocalized with mitochondria. Whole-body fluorescence imaging and immunohistochemical analysis of tumor tissues showed the homing of IL4RPep-1-KLA as well as IL4RPep-1 to KKU-213 tumor in mice. Systemic administration of IL4RPep-1-KLA efficiently inhibited KKU-213 tumor growth, whereas treatment with 5-FU alone did not significantly inhibit tumor growth in mice. No significant systemic side effects including liver toxicity and immunotoxicity were observed in mice during peptide treatments. These findings suggest that IL4RPep-1-KLA holds potential as a targeted therapeutic agent against chemoresistant CCA.

Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity and inhibit tumor progression.

Blockade of programmed cell death ligand-1 (PD-L1) restores T-cell activity and enhances anti-tumor immunity. Screening a phage-displayed peptide library for peptides that selectively bind to PD-L1-overexpressing cells identified two peptides, CLQKTPKQC and CVRARTR (PD-L1Pep-1 and PD-L1Pep-2, respectively) that appeared to block PD-L1. PD-L1Pep-1 and PD-L1Pep-2 preferentially bound to high PD-L1-expressing cells over low PD-L1-expressing cells; binding was further enhanced by interferon-γ, an inducer of PD-L1 expression. Binding affinities of PD-L1Pep-1 and PD-L1Pep-2 were approximately 373 and 281 nM, respectively. Cellular binding of the PD-L1-binding peptides was reduced by silencing PD-L1 gene expression or competition with anti-PD-L1 antibody. PD-L1Pep-1 and PD-L1Pep-2 induced the internalization and downregulated cell surface levels of PD-L1. The PD-L1-binding peptides restored cytokine secretion and T-cell proliferation to cells inhibited by co-culture with tumor cells or culture on PD-L1-coated plates. Intravenously injected PD-L1Pep-1 and PD-L1Pep-2 efficiently homed to tumor tissues, inhibited tumor growth, and increased CD8+/FoxP3+ ratio in mice. The PD-L1-binding peptides in combination with doxorubicin or PD-L1-targeted liposomal doxorubicin inhibited tumor growth and increased CD8+/FoxP3+ ratio more efficiently than doxorubicin alone and untargeted liposomal doxorubicin, respectively. These results suggest that PD-L1Pep-1 and PD-L1Pep-2 block PD-L1 and reinvigorate T-cell activity, inhibiting tumor growth by enhancing anti-tumor immunity.

Peptide-based targeted therapeutics and apoptosis imaging probes for cancer therapy.

Peptides have advantages over antibodies in terms of deep tissue penetration, low immunogenicity, and cost-effective production, but they have short circulation time and poor stability in vivo. Peptides have been extensively used as targeting moieties for the delivery of drug-loaded nanoparticles and function as targeted therapeutics in cancer treatment. Here, we review peptides that are exploited as targeted therapeutics in cancer therapy and apoptosis imaging probes for the monitoring of treatment responses.