Targeting Cancer Cells by Novel Engineered Modular Transporters

Abstract

A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed highaffinity interactions with ErbB1 receptors and A/B-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and Á OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications.

Preparation, cytotoxicity, and in vivo antitumor efficacy of ¹¹¹In-labeled modular nanotransporters

“…Three different MNT molecules were used in these experiments ( Figure 1): 1) melanocortin-1 receptor targeted tDTox-HMP-NLS-MSH (MNT-MSH), M w 70,430 Da, consisting of α-melanocyte-stimulation hormone (MSH) as the ligand module, the diphtheria toxin translocation domain (tDTox) as an endosomolytic module, the optimized nuclear localization sequence of SV-40 large T-antigen (NLS), and hemoglobinlike protein (HMP) as a carrier module; 14 2) epidermal growth factor (EGF) receptor targeted tDTox-HMP-NLS-EGF (MNT-EGF), 13,14 …”

“…13,16,18,26 In this study, we also used the same structure lacking any ligand module to produce MNT with FR targeting. Figure S3.…”

“…We have been working on an alternative strategy for achieving molecularly directed selective delivery of shortrange therapeutics such as Auger electron emitters to malignant cell populations, based on an engineered polypeptide platform. 13 These modular nanotransporters (MNTs) are artificial multifunctional molecules designed to facilitate receptor-specific transport from the cell surface into its nucleus through inclusion of polypeptide domains for accomplishing receptor binding and internalization as well as endosomal escape and nuclear translocation. Moreover, MNTs possess interchangeable component domains, permitting relatively facile exchange of receptor recognition domains, which ultimately might be adapted for different diseases or might help improve homogeneity of drug delivery by targeting several tumor-specific molecules for personalized medicine.…”

“…Moreover, MNTs possess interchangeable component domains, permitting relatively facile exchange of receptor recognition domains, which ultimately might be adapted for different diseases or might help improve homogeneity of drug delivery by targeting several tumor-specific molecules for personalized medicine. MNTs reactive with a variety of tumor-associated receptors have shown promise for delivering short range of action therapeutics including photosensitizers, 13 Ga,17 as they were demonstrated to colocalize in the nuclei of target cancer cells in vitro (up to 60% of internalized found in nuclei) 13,14,16,17 and in vivo, 14 as well as enhance significantly cytotoxicity of all the above-mentioned cargoes in vitro 13,[15][16][17][18] and the therapeutic potential of different photosensitizers in vivo.…”

“…21 All MNTs were synthesized and purified as described earlier. 13,14,18 Briefly, each MNT was expressed in an Escherichia coli suspension (OD 600 0.6-1.0) by the addition of isopropyl-β-d-1-thiogalactopyranoside to a final concentration of 200 µM at 18°C and shaking at 170-180 rpm for 2 h. Bacterial cells were pelleted by centrifugation at 10,000 rpm with a JA-10 rotor at 4°C for 30 min. Then, the pellets were frozen for subsequent lysis in ice-cold 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 5 mg/mL lysozyme, and 0.5% Triton X-100.…”