RESUMO
Mesothelin is an epithelial marker highly expressed at the cell surface of cancer cells from diverse origins, including ovarian and pancreatic adenocarcinomas and mesotheliomas. Previously, we identified and characterized an antimesothelin nanobody (NbG3a) for in vitro diagnostic applications. The main goal of this research was to establish the potential of NbG3a as a molecular imaging agent. Site-specific biotinylated NbG3a (bNbG3a) was bound to streptavidin-conjugated reagents for in vitro and in vivo assays. Initially, we performed microscale thermophoresis to determine the binding affinity between bNbG3a and human ( Kd = 46 ± 8 nM) or mouse ( Kd = 4.8 ± 0.4 nM) mesothelin protein. The human and mouse cross-reactivity was confirmed by in vivo optical imaging using bNbG3a bound to fluorescent streptavidin. We also localized the binding site of nNbG3a on human mesothelin using overlapping peptide scan. NbG3a recognized an epitope within residues 21-65 of the mature membrane bound form of human mesothelin, which is part of the N-terminal region of mesothelin that is important for interactions between mesothelin on peritoneal cells and CA125 on tumor cells. Next, the bNbG3a in vivo half-life after intravenous injection in healthy mice was estimated by ELISA assay to be 5.3 ± 1.3 min. In tumor-bearing animals, fluorescent bNbG3a accumulated in a subcutaneous ovarian xenograft (A1847) and in two syngeneic, orthotopic ovarian tumors (intraovary and intraperitoneal ID8) within an hour of intravenous injection that peaked by 4 h and persisted up to 48 h. MRI analysis of bNbG3a-targeted streptavidin-labeled iron oxides showed that the MRI signal intensity decreased 1 h after injection for a subcutaneous xenograft model of ovarian cancer for bNbG3a-labeled iron oxides compared to unlabeled iron oxides. The signal intensity differences continued up to the final time point at 24 h post injection. Finally, in vivo immunofluorescence 24 or 48 h after bNbG3a intravenous injection showed bNbG3a diffuse distribution of both xenograft and syngeneic ovarian tumors, with local areas of high concentration throughout A1847 human tumor. The data support the use of NbG3a for continued preclinical development and translation to human applications for cancers that overexpress mesothelin.
Assuntos
Reações Cruzadas/imunologia , Proteínas Ligadas por GPI/metabolismo , Neoplasias Ovarianas/patologia , Anticorpos de Domínio Único/imunologia , Animais , Antígeno Ca-125/metabolismo , Linhagem Celular Tumoral , Feminino , Compostos Férricos/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas Ligadas por GPI/imunologia , Xenoenxertos , Humanos , Imageamento por Ressonância Magnética/métodos , Mesotelina , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Estreptavidina/metabolismoRESUMO
Mesothelin, a cancer biomarker overexpressed in tumors of epithelial origin, is a target for nanotechnology-based diagnostic, therapeutic, and prognostic applications. The currently available anti-mesothelin antibodies present limitations, including low penetration due to large size and/or lack of in vivo stability. Single domain antibodies (sdAbs) or nanobodies (Nbs) provide powerful solutions to these specific problems. We generated a phage-display library of Nbs that were amplified from B cells of a llama that was immunized with human recombinant mesothelin. Two nanobodies (Nb A1 and Nb C6) were selected on the basis of affinity (K(D) = 15 and 30 nM, respectively). Nb A1 was further modified by adding either a cysteine to permit maleimide-based bioconjugations or a sequence for the site-specific metabolic addition of a biotin in vivo. Both systems of conjugation (thiol-maleimide and streptavidin/biotin) were used to characterize and validate Nb A1 and to functionalize nanoparticles. We showed that anti-mesothelin Nb A1 could detect native and denatured mesothelin in various diagnostic applications, including flow cytometry, western blotting, immunofluorescence, and optical imaging. In conclusion, anti-mesothelin Nbs are novel, cost-effective, small, and single domain reagents with high affinity and specificity for the tumor-associated antigen mesothelin, which can be simply bioengineered for attachment to nanoparticles or modified surfaces using multiple bioconjugation strategies. These anti-mesothelin Nbs can be useful in both conventional and nanotechnology-based diagnostic, therapeutic and prognostic biomedical applications.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas Ligadas por GPI/imunologia , Nanopartículas/uso terapêutico , Frações Subcelulares/imunologia , Anticorpos Monoclonais/genética , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Células HeLa , Humanos , Mesotelina , Nanopartículas/química , Nanopartículas/ultraestrutura , Engenharia de Proteínas/métodos , Frações Subcelulares/patologiaRESUMO
Nanotechnology is actively being developed for preclinical and clinical oncology applications. Nanoparticle-based immunotargeting against tumor antigens with antibodies or antibody fragments is designed to increase the nanoparticle concentration at the tumor site. However, chemical-based strategies for bioconjugating antibody fragments to nanoparticles typically result in a functionally heterogeneous population of conjugates due to alteration of amino acids within the antigen binding site. The loss of function can be prevented by expressing recombinant antibodies that contain a unique bioconjugation site, which is isolated from the antigen binding site. Biobodies are antibody fragments biosynthetically biotinylated by yeast at a specific biotin acceptor site and secreted into the culture supernatant. The high specificity and affinity between streptavidin-labeled nanoparticles and soluble biobodies allow self-assembly of immunotargeted nanoparticles directly in the yeast culture supernatant. Here, we demonstrate the versatility of biobodies for nanoparticle immunotargeting using streptavidin-labeled superparamagnetic iron oxide nanoparticles as a general, modular scaffold. Biobody-mediated targeting was performed against two antigens (mesothelin and TEM1) that are upregulated in solid tumors. The technology for biosynthetic biotinylation can be extended to proteins other than antibody fragments and adopted by fields outside of oncology for directed modification of any streptavidin-functionalized surface.
Assuntos
Antígenos de Neoplasias/imunologia , Biotina/imunologia , Fragmentos de Imunoglobulinas/imunologia , Nanopartículas/química , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Biotinilação/métodos , Linhagem Celular Tumoral , Meios de Contraste/síntese química , Humanos , Nanopartículas/ultraestruturaRESUMO
Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 +/- 0.11 mM(-1) sec(-1) at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals.
Assuntos
Produtos do Gene tat/síntese química , Espectroscopia de Ressonância Magnética/métodos , Compostos Organometálicos/síntese química , Peptídeos/síntese química , Peptídeos/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Quelantes/química , Meios de Contraste/química , Meios de Contraste/farmacocinética , Relação Dose-Resposta a Droga , Produtos do Gene tat/metabolismo , Compostos Heterocíclicos/química , Humanos , Células Jurkat/metabolismo , Rim/fisiologia , Fígado/fisiologia , Imageamento por Ressonância Magnética/métodos , Mesentério/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Peptídeos/farmacocinética , Distribuição TecidualRESUMO
The metal-binding affinities and three-dimensional structures of three synthetic 18-residue peptides with sequences derived from that of the highly conserved metal-binding motif MXCXXC found in many heavy metal-binding proteins were determined. A change in register of the cysteines and alanines of the sequence from the periplasmic mercury-binding protein, MerP, i.e., CAAC, CACA, and CCAA, affects the specificity of metal binding, in particular, the peptide with vicinal cysteines binds only mercury. The three-dimensional structures of the mercury-bound forms of the three peptides determined in solution by NMR spectroscopy peptides differ considerably, even though they are all linear bicoordinate complexes. The three-dimensional structure of the peptide with CAAC bound to Cd(II) demonstrates that the metal-binding loop is malleable enough to accommodate modes of coordination other than linear bicoordinate.