Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.
Conjugate Characterization - Data and Graphs
This file contains HPLC chromatograms and compiled numerical data concerning the characterization (drug:ab and fluorophore:ab ratios) of conjugates synthesized in this study (Fig 1 and Table 1).
Conjugate Characterization - Linker Stability
This file contains all HPLC chromatograms, graphs and computations for determining antibody-drug linker stability of the fluorescent antibody drug conjugate as described in the paper (Fig 2).
In Vitro Fluorescent Evalution - Quantitation
This file contains all of the raw and analyzed fluorescent quantitation data for assessing localization of conjugates with various cell lines (Fig. 3).
Immunofluorescence Sample Image BxPC-3 CEA 680 PTX
This file is a raw jpeg of an immunofluorescent image used in Fig 3, panel A.
Immunofluorescence Sample Image BxPC-3 CEA 680
This file is a raw jpeg of an immunofluorescent image used in Fig 3, panel A.
Immunofluorescence Sample Image BxPC3 IgG 680 PTX
This file is a raw jpeg of an immunofluorescent image used in Fig 3, panel A.
Immunofluorescence Sample Image BxPC3 IgG 680
This file is a raw jpeg of an immunofluorescent image used in Fig 3, panel A.
In Vitro Fluorescent Evaluation - BxPC3 Flow Data
This file contains the raw flow cytometry data and overlayed images (“analyze” tab) which were arranged for Fig 4.
In Vitro Fluorescent Evaluation - HeLa Flow Data
This file contains the raw flow cytometry data and overlayed images (“analyze” tab) which were arranged for Fig 4.
In Vitro Fluorescent Evaluation - HepG2 Flow Data
This file contains the raw flow cytometry data and overlayed images (“analyze” tab) which were arranged for Fig 4.
In Vitro Fluorescent Evaluation - MCF7 Flow Data
This file contains the raw flow cytometry data and overlayed images (“analyze” tab) which were arranged for Fig 4.
In Vitro Fluorescent Evaluation - CEA Internalization Data
This file contains the compiled and processed data surrounding the quantitative estimation of CEA internalization (Fig 5, panel B).
In Vitro Cytotoxicity Evaluation - MTT Assay Compiled Data
This excel file contains all of the compiled MTT assay data, as well as graphs (Fig 6) and linear regression analysis for estimating IC50 (Table 2).
In Vitro Cytotoxic Evaluation - Flow Cytometry Cell Cycle Analysis
This file contains the raw flow cytometry cell cycle analysis data and analyzed images that were arranged for Fig 7. This file was originally a template that was included with the BD cell cycle analysis kit as described in the paper.
In Vivo Evaluation - Tumor Growth and Auto Exposure Data
This excel file contains all raw tumor growth measurements and graphs for in vivo efficacy evaluation (Fig 8). This file also contains the compiled exposure times generated for Fig 10.
In Vivo Imaging Sample Image - Day 7 - White Light - Raw
This file is a raw jpeg generated for Fig 9. It depicts imaging of a mouse in group 3 imaged under white light as described in the methods section.
In Vivo Imaging Sample Image - Day 7 - Infrared - Raw
This file is a raw jpeg generated for Fig 9. It depicts a mouse in group 3 imaged under an infrared filter set as described in the methods section.
In Vivo Imaging Sample Image - Day 7 - Infrared - False Color
This file is a jpeg generated for Fig 9. It depicts a mouse in group 3 imaged under an infrared filter as described in the methods section. Red false color has been added with Pxlr editor as described in the methods section.
In Vivo Evaluation - ANOVA Analysis
This file contains the statistical analysis of the data generated for Fig 8.