Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide–drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.
Cancer is among the leading causes of death worldwide. According to estimates from the International Agency for Research on Cancer, in 2020, more than 19 million cases and nearly 10 million deaths were reported. Among those, prostate cancer ranked second in terms of incidence, and fifth in terms of deaths caused by cancer globally among men. Moreover, in 112 out of 185 countries, it is the most frequently diagnosed tumour in men. On the other hand, female breast cancer became the leading cause of cancer incidence, surpassing lung cancer, with 11.7% of all cancer cases, and represents the fifth globally leading cause of cancer mortality.
Chemotherapy has always been the most common treatment for cancer. However, the nonspecific distribution of many chemotherapeutics limits their clinical applications and causes high levels of toxicity. Therefore, targeted tumour therapy has appeared as a promising approach to overcome such limitations. Drug delivery systems (DDS) exploit differences between healthy and cancerous cells and tissues, to selectively deliver a toxic payload to the site of action and attempt to minimise off-target side effects. As a result, many antibody–drug conjugates (ADCs) have already obtained FDA and/or EMA approval, such as trastuzumab emtansine (Kadcyla®), brentuximab vedotin (Adcetris®) and, most recently, loncastuximab tesirine (Zylonta®), disitamab vedotin (Aidixi®) and tisotumab vedotin (Tivdak®). Similarly to the emerging concept of ADC technology, peptide–drug conjugates (PDCs) have gained increasing interest due to the distinct benefits of tumour homing peptides. As a matter of fact, having a peptide ligand instead of a monoclonal antibody (mAb) allows us to overcome some of the limitations of ADCs in cancer therapy, such as high production costs, poor tumour permeability and potentially dangerous immune reactions. These advantages recently led to the authorisation of three PDCs for clinical use in cancer: vipivotide tetraxetan, melphalan flufenamide and a somatostatin derivative, 177 Lu-DOTATATE. Several others, such as Bicycle Therapeutics’ BT1718, BT5528 and BT8009, Cybrexa’s CBX-12 and Shenogen’s SNG1005, are undergoing clinical trials.
Choosing an appropriate target is crucial for the development of effective and safe devices for the delivery of chemotherapeutics. For example, tumour cells can be discriminated from normal cells due to upregulated cell surface receptors or enzyme levels. After obtaining encouraging results with GnRH-III-based PDCs directed towards the gonadotropin-releasing hormone receptor (GnRH-R), our research group has focused on the gastrin-releasing peptide receptor (GRP-R, or bombesin receptor 2, BB2), which is overexpressed in several malignancies, such as prostate, breast and lung cancer, while being poorly expressed physiologically in healthy tissues. This receptor is part of the bombesin receptor protein family, together with the neuromedin B receptor (NMB-R, BB1) and the bombesin receptor subtype 3 (BRS-3, BB3). Its native ligand is the gastrin-releasing peptide, of which the C-terminal heptapeptide fragment is common with bombesin (BBN), a 14-mer peptide which was first discovered in the skin of the European fire-bellied toad, Bombina bombina. The mentioned truncated version, BBN (7-14) (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2), maintains the affinity towards GRP-R. Hence, it has been studied as a targeting ligand and has been the starting point for the synthesis of several analogues throughout the years, with retained or improved affinity for GRP-R and increased stability in plasma, but also with modulated agonist or antagonist activity. For example, a very common modification is the insertion of a D-Phe in position 6, reported to increase the affinity. As for position 11, Hoppenz et al. have reported that the presence of β-Ala, together with an Ala in position 13, provides peptides that are highly selective for GRP-R, whereas the selectivity is lost with either Leu 13 or Phe 13 and the binding is lost with D-Ala 13. Moreover, the exchange of Ala 13 to either Aib or N-Me–Ala, conferred longer stability in plasma. The exchange of Leu 13–Met 14 to Sta 13–Leu 14 represents another successful substitution: the affinity towards GRP-R is increased and Sta 13 improves the resistance to the neutral endopeptidase-driven cleavage of the His 12–Leu 13 bond. Furthermore, together with D-Phe 6, it confers an antagonistic activity to the bombesin analogues. Finally, the Gly 11–His 12 bond was reinforced by substituting Gly 11 with N-Me–Gly. Such analogues have been developed and tested for cancer imaging, diagnosis and treatment.
We decided to select some of the described peptides bearing the mentioned substitutions and having affirmed affinity for GRP-R in the low nanomolar range, together with the original BBN (7-14), and use them as targeting moiety to deliver a cytotoxic payload, daunorubicin (Dau), to prostate and breast cancers. Compared to the BBN (7-14) sequence, our targeting peptides are elongated by a D-Phe in position 6 and comprise substitutions in positions 11, 13 and 14. Starting from these variations, we have also generated a new sequence: [D-Phe 6, β-Ala 11, Sta 13, Nle 14]BBN (6-14). The bombesin analogues have been published in the works of different research groups throughout many years, however, a direct comparison between them in terms of drug delivery has never been done.
