Original ArticleDiscovery of peptide drug carrier candidates for targeted multi-drug delivery into prostate cancer cells
Introduction
Metastatic castration-resistant prostate cancer (mCRPC) is essentially incurable [1] and in 2014 the disease accounted for approximately 30,000 deaths in the US alone [2]. Androgen deprivation therapy, for many years the standard first-line treatment for mCRPC is initially effective in most patients, but disease inevitably progresses [3]. Newer molecular targeted therapies have shown clinical benefit for patients with mCRPC including chemotherapies, immunotherapies and radiopharmaceuticals. However it is not yet clear how to sequence these therapies in individual patients to achieve long-term clinical efficacy with low toxicity, nor is it clearly understood how cross-resistance to each component develops (reviewed in Ref. [3]. The continual genetic evolution of prostate cancer (PrC) cells is an additional complication [4], requiring repeated patient monitoring to update chemotherapy regimens [3], a process of “cat-and-mouse” that is expensive and carries implications for drug toxicity, patient morbidity and survival.
Targeted Drug Delivery (TDD) systems based for example on antibodies [5] and peptides [6]may overcome many of the limitations of current mCRPC therapies. For example our own studies demonstrate that attachment of a small ligand- or peptide-cancer cell targeting agent to cytotoxic drugs affords several advantages that include: specific delivery of drug to the target cell, regulated cytotoxicity, bypassing of drug resistance, reversal of drug resistance, delivery of higher drug payloads and simultaneous delivery of cytotoxic drugs with different mechanisms of action [7], [8], [9], [10], [11], [12].
TDDs assume the presence of an appropriate cell surface target molecule and one of the most studied mCRPC related cell surface components is Prostate Specific Membrane Antigen (PSMA), a 100 KDa type II transmembrane glycosylated protein. PSMA is very commonly expressed on PrC cells. The biology and clinical relevance of PMSA expression has been extensively researched and it has been targeted by a diverse selection of small ligands, antibodies and peptides to which have been conjugated a variety of radiolabels and drugs [13], [14]. Some of these constructs have been evaluated clinically (some are still ongoing, see www.clinicaltrials.gov) and FDA-approved PSMA-targeted products are available for diagnostic imaging [15]. Despite these advances, many questions remain about the efficacy of PSMA-targeted therapeutics [13], [14], [15].
The most clinically advanced peptide-based TDD systems are bioconjugates incorporating analogues of the natural hormone Gonadotropin-Releasing Hormone (GnRH) (reviewed in Ref. [16]) that target the GnRH receptor. However, this approach has several limitations. a) GnRH receptors are also present on a variety of normal tissues. b) Due to the structure of the receptors, internalization of the bioconjugate is relatively slow; c) It has proven very difficult to prepare active conjugates of these analogues with drugs other that anthracyclines such as doxorubicin [16].
To expand the spectrum of targeting peptides for TDD, phage display technology is being used [17], [18]. Jayanna et al. [19] and also Fagbohun et al. [20] used phage display to isolate clones that were internalized by PrC cells, with the aim of using the phage themselves, not the isolated peptides, as delivery vehicles. The Arap group [21]recently described phage clones internalized by prostate cancer cells in bone metastases, however activity with isolated peptides was not described. Other studies focused on using phage libraries to isolate peptides binding other known receptors on PrC cells [22], [23], [24], [25]. However, the peptides were mainly designed to inhibit the activity of a receptor.
Our goal was different. We set out to use phage display peptide libraries to isolate novel PrC targeting peptides for use in Peptide-Drug-Conjugates (PDCs) that would be effective in the treatment of mCRPC. Our approach was to use unbiased exposure of the phage library to mCRPC cells, without selecting for peptides that bound a pre-defined receptor. We used strict criteria to select appropriate phage clones and their displayed peptides: a) internalization of the phage into the target cell, b) target cell specificity c) uptake of the synthesized display peptide into target cells d) accumulation of the peptide into tumor and e) incorporation of the peptide into both mono-drug and dual-drug-PDCs that killed PrC cells.
We compared a commercial phage library expressing 7-mer linear peptides with the same library after it had been injected into a normal mouse. Phage clones recovered from the bloodstream of the mouse were assumed not to have effective affinity for normal tissues and were not degraded by the liver. This manipulation significantly reduced the number of clones in the library, thus increasing the probability of “fishing out” appropriate on-target clones. Indeed the two peptides (P10 and P12) that we have so far tested most thoroughly and are reporting on here were both derived from the second library. These peptides are active on a range of metastatic prostate cancer cell lines but not on normal cells. Importantly, the preparation of active peptide-dye conjugates was found to be terminus dependent and this guided the synthesis of PDCs that were cytotoxic to PrC target cells. These peptides are now moving into an advanced program of pre-clinical testing as a potential novel targeted drug therapy for mCRPC.
Section snippets
Cells and reagents
The cell lines used in this study represented malignant prostate cancer (PC-3, LNCaP, DU-145), cervical cancer (HeLa) and human embryonic kidney cells (HEK-293). All cells were obtained from the ATCC. PC-3, DU-145 and LNCaP were grown in RPMI 1640 (Thermo Fischer Scientific, Waltham, Ma., USA). HeLa were grown in MEM Alpha medium and HEK-293 in DMEM (Thermo Fischer). All media were supplemented with 10% FBS (Thermo Fischer), 3 mM l-Glutamine and antibiotics (Biological Industries, Bet Haemek,
PC-3 cells internalize phage
Both the parent Ph.D-7 phage library (L1) and the in vivo absorbed (L2) phage library were exposed to PC-3 and internalized phage were retrieved. After titration, phage from 15 individual plaques were recovered. Sequencing of the peptide inserts showed that only one peptide sequence was obtained from each plaque, indicating the clonality of the phage in each plaque. Of the plaques isolated from the original (L1) library (Table 1), 2 displayed the same sequence, another two displayed a different
Discussion
Our study describes new approaches to utilizing phage display peptide libraries for developing Targeted Drug Delivery systems for prostate cancer therapy. First, instead of targeting classical prostate cancer surface markers such as PSMA [23], or GnRH receptors [16] we used a screening strategy that favors the discovery of novel peptides that target potentially other cell surface components that would serve as appropriate ports of drug delivery into the cell. Second, for biological (lack of
Funding sources
Ariel University Research Authority Grant # RA1500662.
Ariel R&D Tech Transfer Grant #AC1500061.
OB and BR were supported by graduate student stipends from Ariel University.
Acknowledgements
The authors wish to thank Lena Tuchinsky for her expert assistance with the confocal microscopy and the animal studies. The research was funded by the Ariel University Research Authority and the Ariel University Technology Transfer Office (Ariel R&D Ltd).
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