ABSTRACT
We report combined therapy using upconversion nanoparticles (UCNP) coupled to two therapeutic agents: beta-emitting radionuclide yttrium-90 (90Y) fractionally substituting yttrium in UCNP, and a fragment of the exotoxin A derived from Pseudomonas aeruginosa genetically fused with a targeting designed ankyrin repeat protein (DARPin) specific to HER2 receptors. The resultant hybrid complex UCNP-R-T was tested using human breast adenocarcinoma cells SK-BR-3 overexpressing HER2 receptors and immunodeficient mice, bearing HER2-positive xenograft tumors. The photophysical properties of UCNPs enabled background-free imaging of the UCNP-R-T distribution in cells and animals. Specific binding and uptake of UCNP complexes in SK-BR-3 cells was observed, with separate 90Y- and PE40-induced cytotoxic effects characterized by IC50 140 µg/mL (UCNP-R) and 5.2 µg/mL (UCNP-T), respectively. When both therapeutic agents were combined into UCNP-R-T, the synergetic effect increased markedly, â¼2200-fold, resulting in IC50 = 0.0024 µg/mL. The combined therapy with UCNP-R-T was demonstrated in vivo.
Subject(s)
Drug Delivery Systems/methods , Endotoxins/therapeutic use , Nanoparticles/therapeutic use , Nanotechnology/methods , Neoplasms/therapy , Radiotherapy/methods , Adenocarcinoma/therapy , Ankyrin Repeat , Breast Neoplasms/therapy , Cell Line, Tumor , Female , Humans , Neoplasms/diagnostic imaging , Pseudomonas aeruginosa , Radionuclide Imaging/methods , Receptor, ErbB-2/metabolism , Recombinant Proteins , Yttrium Radioisotopes/therapeutic useABSTRACT
Tumor-targeted delivery of cytotoxins presents considerable advantages over their passive transport. Chemical conjugation of cytotoxic module to antibody is limited due to insufficient reproducibility of synthesis, and recombinant immunotoxins are aimed to overcome this disadvantage. We obtained genetically encoded immunophotosensitizer 4D5scFv-miniSOG and evaluated its photocytotoxic effect in vitro. A single-chain variable fragment (scFv) of humanized 4D5 antibody was used as a targeting vehicle for selective recognition of the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) overexpressed in many human carcinomas. As a phototoxic module we used a recently described photoactivated fluorescent flavoprotein miniSOG. We found that recombinant protein 4D5scFv-miniSOG exerts a highly specific photo-induced cytotoxic effect on HER2/neu-positive human breast adenocarcinoma SK-BR-3 cells (IC50= 160 nM). We demonstrated that the 4D5scFv-miniSOG specifically binds to HER2-positive cells and internalizes via receptor-mediated endocytosis. Co-treatment of breast cancer cells with 4D5scFv-miniSOG and Taxol or junction opener protein JO-1 produced remarkable additive effects.
Subject(s)
Antineoplastic Agents/pharmacology , Flavoproteins/pharmacology , Immunotoxins/pharmacology , Molecular Targeted Therapy/methods , Neoplasms/therapy , Photosensitizing Agents/pharmacology , Recombinant Fusion Proteins/pharmacology , Single-Chain Antibodies/pharmacology , Cell Line, Tumor , Cell Proliferation , Cell Survival , Flavoproteins/genetics , Humans , Immunotoxins/genetics , Inhibitory Concentration 50 , Light , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Receptor, ErbB-2/metabolism , Recombinant Fusion Proteins/genetics , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolismABSTRACT
Myelopeptide-2 (MP-2; Leu-Val-Val-Tyr-Pro-Trp), originally isolated from the supernatant of porcine bone marrow cell culture, is able to restore the mitogen responsiveness of human T lymphocytes inhibited by conditioned medium from HL-60 leukemia cells or measles virus. This effect is based on the ability of MP-2 to recover the reduced interleukin (IL)-2 synthesis and IL-2 receptor (IL-2R) expression in human T lymphocytes treated with these harmful agents. The involvement of other cytokines in MP-2 restoration of the reduced IL-2 synthesis in T lymphocytes is experimentally studied. It is shown that T helper (TH) 1 and TH2 cytokines are acting in close interaction, the character of which depends on the immune status of the T-lymphocyte donors. The data obtained allow one to suggest that the MP-2 involvement in regulatory processes is directed to the maintenance of immune homeostasis. This peptide is perspective to be applied in antitumor and antivirus therapy.
