RESUMO
The tumor vasculature and extracellular matrix make attractive targets for distinguishing solid tumors from normal cells. In solid tumors, the processes of angiogenesis and metastasis potentially give rise to unique epitopes not usually accessible in homeostatic organs. Specific targeting of solid tumors for radioimmunotherapy requires that the targeting agent accumulate rapidly and at high levels at the tumor site. This study involved the selection of scFvs that recognize laminin-1 in vitro from the Tomlinson I and J phage display libraries. Selected, purified scFvs were radioiodinated and injected in tumor-bearing mice. One of these, scFv 15-9, exhibited preferential accumulation at subcutaneous tumors when compared to other antilaminin scFvs or to a control scFv. Autoradiographic analysis indicated that scFv15- 9 also displayed a higher vessel:parenchyma ratio than did two other antilaminin scFvs, scFv 15-6 and scFv 15-1, indicating a preferential accumulation of scFv 15-9 around vessel structures. Immunohistochemistry confirmed that scFv 15-9 accumulated at sites of endothelial cells lining vessel structures where significant levels of laminin were present. These data demonstrate that scFv 15-9 binds to a specific epitope on laminin and has potential for tumor endoradiotherapy in subcutaneous tumors.
Assuntos
Laminina/química , Neoplasias/imunologia , Radioimunoterapia/métodos , Animais , Especificidade de Anticorpos , Membrana Basal , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos , Matriz Extracelular/metabolismo , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Imuno-Histoquímica , Imunoterapia/métodos , Laminina/imunologia , Camundongos , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Neovascularização Patológica , Biblioteca de Peptídeos , Ligação Proteica , Distribuição TecidualRESUMO
A human scFv, 15-9, was selected from a phage display library for binding to murine laminin-1. A diabody was made from the scFv by shortening the linker from 15 to 5 amino acids between the VH and VL sequence. Radioiodinated scFv and diabody were analyzed for size, binding to laminin, and biodistribution in tumor bearing mice. Diabody preparations at concentrations greater than 10 nM were largely dimer forms (approximately 60 kDa) as judged by gel filtration, but diluted diabody was eluted as a monomer (approximately 30 kDa). At low concentrations the radiolabeled diabody did not bind well to laminin. The (125)I diabody had significantly lower accumulation in tumors than did the scFv when injected at lower concentrations. These data indicate that the diabody dimer dissociates at concentrations of about 10nM resulting in monomers with no binding activity for laminin and poor tumor homing properties.
Assuntos
Afinidade de Anticorpos/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Neoplasias/imunologia , Engenharia de Proteínas , Subunidades Proteicas/química , Animais , Especificidade de Anticorpos/imunologia , Cromatografia em Gel , Feminino , Região Variável de Imunoglobulina/análise , Região Variável de Imunoglobulina/genética , Radioisótopos do Iodo , Laminina/imunologia , Laminina/metabolismo , Camundongos , Camundongos SCID , Transplante de Neoplasias , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Subunidades Proteicas/farmacocinética , Distribuição TecidualRESUMO
Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.
Assuntos
Reagentes de Ligações Cruzadas/química , Peptídeos/análise , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Avidina/metabolismo , Bovinos , Cromatografia de Afinidade , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Análise de Sequência de ProteínaRESUMO
A mouse monoclonal antibody (MAb-9) produced by immunization with a human esophageal carcinoma cell line, TE-2 (derived from undifferentiated squamous cell carcinoma) reacted specifically with about 30% of esophageal carcinoma cell lines and tissue sections from clinical samples. MAb-9 showed minimal reactivity with normal esophageal tissue. (125)I, fluorescent or gold particle labeled MAb-9 bound to TE-2 cell surfaces. (125)I-radiolabeled MAb-9 was used to detect reactive material from cell extracts in Western blot. Treatment of TE-2 membrane proteins with neuraminidase, N-glycanase or O-glycanase reduced antigen detection. Treatment of cells with periodic acid destroyed antibody binding in ELISA. Lipid extracts from cell membranes, containing glycolipids, also reacted with MAb-9. MAb-9 was used to purify target antigen from detergent solubilized membrane proteins and the prominent bands from subsequent gel electrophoresis were trypsin digested and analyzed by mass spectrometry. Peptides from alpha(3) and beta(1) integrin chains were identified. These data indicate that alpha3beta1integrin is prominently expressed on certain esophageal carcinomas and that a specific carbohydrate unit is selectively displayed on the alpha(3) integrin subunit as well as on glycolipid on the cell surface. The alpha3beta1 integrin expressed on A-431 carcinoma cells does not display this carbohydrate epitope and is not detected by MAb-9. Thus, expression of the carbohydrate epitope is the basis for the tumor selective reaction of MAb-9 with a subset of esophageal carcinomas.