RESUMO
Sensitive quantification of the pharmacokinetics of poly(ethylene glycol) (PEG) and PEGylated molecules is critical for PEGylated drug development. Here, we developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for PEG by tethering an anti-PEG antibody (AGP3) via tethers with different dimensions on the surface of 293T cells (293T/S-αPEG, short-type cells; 293T/L-αPEG, long-type cells; 293T/SL-αPEG, hybrid-type cells) to improve the binding capacity and detection limit for free PEG and PEGylated molecules. The binding capacity of hybrid-type cells for PEG-like molecules (CH3-PEG5K-FITC (FITC = fluorescein isothiocyanate) and eight-arm PEG20K-FITC) was at least 10-80-fold greater than that of 293T cells expressing anti-PEG antibodies with uniform tether lengths. The detection limit of free PEG (OH-PEG3K-NH2 and CH3-PEG5K-NH2) and PEG-like molecule (CH3-PEG5K-FITC, CH3-PEG5K-SHPP, and CH3-PEG5K-NIR797) was14-137 ng mL-1 in the hybrid-type cell-based sandwich ELISA. 293T/SL-αPEG cells also had significantly higher sensitivity for quantification of a PEGylated protein (PegIntron) and multiarm PEG macromolecules (eight-arm PEG20K-NH2 and eight-arm PEG40K-NH2) at 3.2, 16, and 16 ng mL-1, respectively. Additionally, the overall binding capacity of 293T/SL-αPEG cells for PEGylated macromolecules was higher than that of 293T/S-αPEG or 293T/L-αPEG cells. Anchoring anti-PEG antibodies on cells via variable-length tethers for cell-based sandwich ELISA, therefore, provides a sensitive, high-capacity method for quantifying free PEG and PEGylated molecules.
Assuntos
Anticorpos/metabolismo , Membranas/metabolismo , Polietilenoglicóis/análise , Reagentes de Ligações Cruzadas/química , Ensaio de Imunoadsorção Enzimática , Células HEK293 , HumanosRESUMO
Attachment of poly(ethylene glycol) to proteins can mask immune epitopes to increase serum half-life, reduce immunogenicity, and enhance in vivo biological efficacy. However, PEGylation mediated epitope-masking may also limit sensitivity and accuracy of traditional ELISA. We previously described an anti-PEG-based sandwich ELISA for universal assay of PEGylated molecules. Here, we compared the quantitative assessment of PEGylated interferons by anti-PEG and traditional anti-interferon sandwich ELISA. The detection limits for PEG-Intron (12k-PEG) and Pegasys (40k-PEG) were 1.9 and 0.03 ng/mL for anti-PEG ELISA compared to 0.18 and 0.42 ng/mL for traditional anti-interferon sandwich ELISA. These results indicate that the anti-PEG sandwich ELISA was insensitive to PEGylation mediated epitope-masking and the sensitivity increased in proportion to the length of PEG. By contrast, PEG-masking interfered with detection by traditional anti-interferon sandwich ELISA. Human and mouse serum did not affect the sensitivity of anti-PEG ELISA but impeded traditional anti-interferon sandwich ELISA. The anti-PEG sandwich ELISA was comparable to anti-interferon sandwich ELISA and radioassay of 131I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.
Assuntos
Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Interferons/análise , Interferons/química , Polietilenoglicóis/química , Animais , Feminino , Humanos , Interferons/sangue , Camundongos , Polietilenoglicóis/farmacocinéticaRESUMO
Poly(ethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/alphaPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alphaPEG cell-coated plate bound biotinylated PEG(5K) (CH(3)-PEG(5K)-biotin) and CH(3)-PEG(5K)-(131)I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5, 10, or 20 kDa) and a known amount of CH(3)-PEG(5K)-biotin allowed construction of a reproducible competition curve. The alphaPEG cell-based competition ELISA measured small molecules derivatized by PEG(2K), PEG(5K), PEG(10K), PEG(20K), and PEG(5K) at concentrations as low as 58.6, 14.6, 3.7, 3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the alphaPEG cell-based competition ELISA. Finally, we show here that the alphaPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH(3)-PEG(5K)-(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.
Assuntos
Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Polietilenoglicóis/análise , Polietilenoglicóis/farmacocinética , Animais , Células 3T3 BALB , Bovinos , Feminino , Camundongos , Soro/química , Soroalbumina Bovina/químicaRESUMO
For those patients with HER2-overexpressing breast cancer, treatment with PEGylated liposomal doxorubicin (PLD) is inefficacious due to the intrinsic low sensitivity to doxorubicin. A very large increase in drug accumulation by active targeting may enhance the therapeutic efficacy of PLD. We established a humanized bispecific antibody (BsAb; mPEG × HER2) which has dual specificity for methoxy-polyethylene glycol (mPEG) and human epidermal growth factor receptor 2 (HER2) to enhance the specificity, internalization and anticancer activity of PLD for cancer cells that overexpress HER2. One-step formulation of PLD with mPEG × HER2 converted the PLD into HER2 targeted liposomes that were stable at 4 °C in PBS as well as at 37 °C in the presence of serum. αHER2/PLD induced receptor-mediated endocytosis and enhanced doxorubicin accumulation in MCF7/HER2 (HER2-amplified) breast cancer cells. αHER2/PLD also displayed more than 200-fold increased cytotoxicity to MCF7/HER2 cells and 28-fold increased cytotoxicity to drug-resistant MDA-MB-361 cells with a physical deletion of the TOP2A gene. αHER2/PLD specifically accumulated doxorubicin in the nucleus of cancer cells in tumor-bearing mice and produced significantly greater antitumor activity against MCF7/HER2 (P < 0.0001) and MDA-MB-361 (P < 0.05) tumors as compared to untargeted PLD. Furthermore, the cardiotoxicity of αHER2/PLD was similar to that of PLD in human cardiomyocytes and in mice. Our results indicate that the one-step formulation of PLD by mPEG × HER2 is a simple method to confer tumor specificity, increase drug internalization and enhance the anticancer activity of PLD against HER2-overexpressing and doxorubicin-resistant breast cancer.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Nanopartículas/química , Polietilenoglicóis/química , Animais , Antineoplásicos/química , Transporte Biológico , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Composição de Medicamentos , Humanos , Células MCF-7 , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismoRESUMO
Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR(+) and HER2(+) cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drug-loaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Doxorrubicina/análogos & derivados , Nanopartículas , Polietilenoglicóis , Animais , Antibióticos Antineoplásicos/uso terapêutico , Anticorpos Biespecíficos/química , Anticorpos Monoclonais Humanizados/química , Células 3T3 BALB , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Receptores ErbB/análise , Feminino , Humanos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Imagem Óptica , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/uso terapêutico , Receptor ErbB-2/análiseRESUMO
PURPOSE: To develop an imaging technology for protease activities in patients that could help in prognosis prediction and in design of personalized, protease-based inhibitors and prodrugs for targeted therapy. EXPERIMENTAL DESIGN: Polyethylene glycol (PEG) was covalently attached to the N-terminus of a hydrophilic peptide substrate (GPLGVR) for matrix metalloproteinase (MMP) to increase hydrophilicity. PEG-peptide was then linked to a hydrophobic tetramethylrhodamine (TMR) domain and labeled with (18)F to form a PEG-peptide-(18)F-TMR probe. Specific cleavage of the probe by MMP2 was tested in vitro by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF). In vivo imaging of MMP2-expressing tumors was evaluated by micro-PET. RESULTS: The hydrophobic TMR fragment (948 Da) was specifically generated by MMP2 enzymes and MMP-expressing HT1080 cells but not control MCF-7 cells. MMP-expressing HT1080 cells and tumors selectively accumulated the hydrolyzed, hydrophobic TMR fragment at sites of protease activity. Importantly, we found that (18)F-labeled probe ((18)F-TMR) preferentially localized in HT1080 tumors but not control MCF-7 tumors as shown by micro-PET. Uptake of the probe in HT1080 tumors was 18.4 ± 1.9-fold greater than in the MCF-7 tumors 30 minutes after injection. These results suggest that the PEG-peptide-(18)F-TMR probe displays high selectivity for imaging MMP activity. CONCLUSIONS: This strategy successfully images MMP expression in vivo and may be extended to other proteases to predict patient prognosis and to design personalized, protease-based inhibitors and prodrug-targeted therapies.