Peptide-Drug Conjugate Targeting Keratin 1 Inhibits Triple-Negative Breast Cancer in Mice.

Selective delivery of chemotherapy to the tumor site while sparing healthy cells and tissues is an attractive approach for cancer treatment. Carriers such as peptides can facilitate selective tumor targeting and payload delivery. Peptides with specific affinity for the overexpressed cell-surface receptors in cancer cells are conjugated to chemotherapy to afford peptide-drug conjugates (PDCs) that show selective uptake by cancer cells. Using a 10-mer linear peptide (WxEAAYQrFL) called 18-4 that targets and binds breast cancer cells, we designed a peptide 18-4-doxorubicin (Dox) conjugate with high specific toxicity toward triple-negative breast cancer (TNBC) MDA-MB-231 cells and 30-fold lower toxicity to normal breast MCF10A epithelial cells. Here, we elucidate the in vivo activity of this potent and tumor-selective peptide 18-4-Dox conjugate in mice bearing orthotopic MDA-MB-231 tumors. Mice treated with four weekly injections of the conjugate showed significantly lower tumor volumes compared to mice treated with free Dox at an equivalent Dox dose. Immunohistochemical (IHC) analysis of mice tissues revealed that treatment with a low dose of PDC (2.5 mg/kg of Dox equiv) reduced the expression of proliferation markers (PCNA and Ki-67) and increased apoptosis (evidenced by increased caspase-3 expression). At the same dose of free Dox (2.5 mg/kg), the expression of these markers was similar to that of saline treatment. Accordingly, significantly more Dox accumulated in tumors of conjugate-treated mice (7-fold) compared to the Dox-treated mice, while lower levels of Dox were observed in the liver, heart, and lungs of peptide-Dox conjugate-treated mice (up to 3-fold less) than Dox-treated mice. The IHC analysis of keratin 1 (K1), the receptor for peptide 18-4, revealed K1 upregulation in tumors and low levels in normal mammary fat pad and liver tissues from mice, suggesting preferential uptake of PDCs by TNBC to be K1 receptor-mediated. Taken together, our data support the use of a PDC approach to deliver chemotherapy selectively to the TNBC to inhibit tumor growth.

Targeting Triple Negative Breast Cancer Cells with Novel Cytotoxic Peptide-Doxorubicin Conjugates.

In this study, we have designed and synthesized two novel peptide-drug conjugates (PDCs) where the drug, doxorubicin (Dox), is linked to the peptide via a succinimidyl thioether bond or a hydrazone linker. A highly specific and proteolytically stable breast cancer cell targeting peptide (WxEAAYQrFL) is conjugated to Dox to synthesize peptide-Dox thioether (1) or hydrazone (2) conjugate. The evaluation of the stability in water, media, and human serum showed that the conjugate 1 with the succinimidyl thioether linkage is more stable compared to the acid-sensitive hydrazone containing conjugate 2. The cytotoxicity studies showed that the two PDCs were as toxic as free Dox toward the triple negative breast cancer (TNBC) cells and were 7-30 times less toxic (IC50 1.2-4.7 µM for TNBC cells versus 15-39 µM for noncancerous cells) toward the noncancerous breast cells compared to the free doxorubicin (IC50 0.35-1.5 µM for TNBC cells versus 0.24 µM for noncancerous cells). The results from the comparative study of the two PDCs suggest that both may have translational potential for TNBC treatment.