RESUMEN
Protease-cleavable supramolecular oligopeptide nanofilaments are promising materials for targeted therapeutics and diagnostics. In these systems, single amino acid substitutions can have profound effects on the supramolecular structure and consequent proteolytic degradation, which are critical parameters for their intended applications. Herein, we describe changes to the self-assembly and proteolytic cleavage of iodine containing sequences for future translation into matrix metalloprotease (MMP-9)-activated supramolecular radio-imaging probes. We use a systematic single amino acid exchange in the tripeptide linker region of these peptide amphiphiles to provide insights into the role of each residue in the supramolecular assemblies. These modifications resulted in dramatic changes in the nature of the assembled structures formed, including an unexpected chiral inversion. By using circular dichroism, atomic force microscopy, Fourier transform infrared spectroscopy, and molecular dynamics simulations, we found that the GD loop, a common motif in ß-turn elements, induced a reversal of the chiral orientation of the assembled nanofibers. In addition to the impact on peptide packing and chirality, MMP-9-catalyzed hydrolysis was evaluated for the four peptides, with the ß-sheet content found to be a stronger determinant of enzymatic hydrolysis than supramolecular chirality. These observations provide fundamental insights into the sequence design in protease cleavable amphiphilic peptides with the potential for radio-labeling and selective biomedical applications.
Asunto(s)
Metaloproteinasa 9 de la Matriz , Nanofibras , Péptidos/química , Aminoácidos/química , Nanofibras/química , Péptido HidrolasasRESUMEN
Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a 177Lu-labeled radioimmunoconjugate based on HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-Aâ³-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [177Lu]Lu3+ to produce the completed radioimmunoconjugate, [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/µmol. [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD to that of a 177Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD matched that of 177Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.
Asunto(s)
Neoplasias de la Mama , Inmunoconjugados , Humanos , Animales , Ratones , Femenino , Inmunoconjugados/uso terapéutico , Línea Celular Tumoral , Trastuzumab/uso terapéutico , Trastuzumab/farmacocinética , Quelantes/química , Neoplasias de la Mama/tratamiento farmacológico , Ácido Pentético/química , Inmunoglobulina G/uso terapéuticoRESUMEN
Matrix metalloproteinase (MMP) enzymes are over-expressed by some metastatic cancers, in which they are responsible for the degradation and remodeling of the extracellular matrix. In recent years, MMPs have emerged as promising targets for enzyme-responsive diagnostic probes because oligopeptides can be designed to be selectively hydrolyzed by exposure to these enzymes. With the ultimate goal of developing radio-iodinated peptides as supramolecular building blocks for MMP-sensitive tools for nuclear imaging and therapy, we designed three MMP-9-responsive peptides containing either tyrosine or iodotyrosine to assess the impact of iodotyrosine introduction to the peptide structure and cleavage kinetics. We found that the peptides containing iodotyrosine underwent more rapid and more complete hydrolysis by MMP-9. While the peptides under investigation were predominantly disordered, it was found that iodination increased the degree of aromatic residue-driven aggregation of the peptides. We determined that these iodination-related trends stem from the improved overall intramolecular order through H- and halogen bonding, in addition to intermolecular organization of the self-assembled peptides due to steric and electrostatic effects introduced by the halogenated tyrosine. These fundamental observations provide insights for the development of enzyme-triggered peptide aggregation tools for localized radioactive iodine-based tumor imaging.
Asunto(s)
Metaloproteinasa 9 de la Matriz , Neoplasias de la Tiroides , Halogenación , Humanos , Radioisótopos de Yodo , Cinética , Metaloproteinasa 9 de la Matriz/metabolismo , Péptidos/química , Péptidos/metabolismo , Tirosina/metabolismoRESUMEN
Metallic radionuclides have been instrumental in the field of nuclear imaging for over half a century. While recent years have played witness to a dramatic rise in the use of radiometals as labels for chelator-bearing biomolecules, imaging agents based solely on coordination compounds of radiometals have long played a critical role in the discipline as well. In this work, we seek to provide a brief overview of metal complex-based radiopharmaceuticals for positron emission tomography (PET) and single photon emission computed tomography (SPECT). More specifically, we have focused on imaging agents in which the metal complex itself rather than a pendant biomolecule or targeting moiety is responsible for the in vivo behavior of the tracer. This family of compounds contains metal complexes based on an array of different nuclides as well as probes that have been used for the imaging of a variety of pathologies, including infection, inflammation, cancer, and heart disease. Indeed, two of the defining traits of transition metal complexes-modularity and redox chemistry-have both been creatively leveraged in the development of imaging agents. In light of our audience, particular attention is paid to structure and mechanism, though clinical data is addressed as well. Ultimately, it is our hope that this review will not only educate readers about some of the seminal work performed in this space over the last 30 years but also spur renewed interest in the creation of radiopharmaceuticals based on small metal complexes.