ABSTRACT
PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. METHODS: Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 (6)-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. RESULTS: The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 +/- 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 +/- 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 +/- 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. CONCLUSION: The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.
Subject(s)
ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Molecular Imaging/methods , Optical Phenomena , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/metabolism , Alkyl and Aryl Transferases/genetics , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic , Coloring Agents/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Protein Transport , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Time FactorsABSTRACT
Death-associated protein kinase 2 (DAPK2) is a calcium/calmodulin-regulated proapoptotic serine/threonine kinase that acts as a tumor suppressor. Here we show that DAPK2 is down-regulated in Hodgkin lymphoma-derived tumor cell lines and that promoter-region hypermethylation is one mechanism for DAPK2 inactivation. To determine whether selective reconstitution of DAPK2 catalytic activity in these cells could induce apoptosis, we created a fusion protein comprising a human CD30 ligand conjugated to a human DAPK2 calmodulin-deletion mutant. Thus, recombinant immunokinase DAPK2'-CD30L has a constitutive kinase activity with enhanced proapoptotic function. We show that this immunokinase fusion protein inhibits cell proliferation and induces apoptotic cell death specifically in CD30/DAPK2-negative tumor cell lines. This proof-of-concept study provides the first demonstration of therapeutic strategies based on the restoration of a defective, tumor-suppressing kinase activity by a novel class of recombinant immunotherapeutics.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , CD30 Ligand/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Hodgkin Disease/immunology , Immunotherapy , Recombinant Fusion Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Apoptosis/genetics , Apoptosis/immunology , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , CD30 Ligand/genetics , CD30 Ligand/immunology , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/immunology , Cell Line, Tumor , Cell Proliferation , DNA Methylation , Death-Associated Protein Kinases , Down-Regulation , Gene Expression Regulation, Enzymologic , Hodgkin Disease/enzymology , Hodgkin Disease/genetics , Hodgkin Disease/pathology , Hodgkin Disease/therapy , Humans , Protein Engineering , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/immunology , U937 CellsABSTRACT
Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-Tag (1-3) . Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is site-directed, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-Tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, followed by analysis by flow cytometry and confocal microscopy. All ligands retained their original antigen-binding properties when fused to the SNAP-Tag. The combination of recombinant antibodies or protein ligands with the SNAP-Tag facilitates simple and efficient covalent modification with a broad range of substrates, thus providing a useful and advantageous alternative to existing coupling strategies.
