Abstract
Imaging of intranuclear epitopes using antibodies tagged to cell-penetrating peptides has great potential given its versatility, specificity, and sensitivity. However, this process is technically challenging because of the location of the target. Previous research has demonstrated a variety of intranuclear epitopes that can be targeted with antibody-based radioimmunoconjugates. Here, we developed a controlled-expression model of nucleus-localized green fluorescent protein (GFP) to interrogate the technical limitations of intranuclear SPECT using radioimmunoconjugates, notably the lower target-abundance detection threshold. Methods: We stably transfected the lung adenocarcinoma cell line H1299 with an enhanced GFP (EGFP)-tagged histone 2B (H2B) and generated 4 cell lines expressing increasing levels of GFP. EGFP levels were quantified using Western blot, flow cytometry, and enzyme-linked immunosorbent assay. An anti-GFP antibody (GFP-G1) was modified using dibenzocyclooctyne-N3-based strain-promoted azide-alkyne cycloaddition with the cell-penetrating peptide TAT (GRKKRRQRRRPPQGYG), which also includes a nuclear localization sequence, and the metal ion chelator N3-Bn-diethylenetriamine pentaacetate (DTPA) to allow radiolabeling with 111In. Cell uptake of 111In-GFP-G1-TAT was evaluated across 5 cell clones expressing different levels of H2B-EGFP invitro. Tumor uptake in xenograft-bearing mice was quantified to determine the smallest amount of target epitope that could be detected using 111In-GFP-G1-TAT. Results: We generated 4 H1299 cell clones expressing different levels of H2B-EGFP (0-1 million copies per cell, including wild-type H1299 cells). GFP-G1 monoclonal antibody was produced and purified in house, and selective binding to H2B-EGFP was confirmed. The affinity (dissociation constant) of GFP-G1 was determined as 9.1 ± 3.0 nM. GFP-G1 was conjugated to TAT and DTPA. 111In-GFP-G1-TAT uptake in H2B-EGFP-expressing cell clones correlated linearly with H2B-EGFP expression (P < 0.001). In vivo xenograft studies demonstrated that 111In-GFP-G1-TAT uptake in tumor tissue correlated linearly with expression of H2B-EGFP (P = 0.004) and suggested a lower target-abundance detection threshold of approximately 240,000 copies per cell. Conclusion: Here, we present a proof-of-concept demonstration that antibody-based imaging of intranuclear targets is capable both of detecting the presence of an epitope of interest with a copy number above 240,000 copies per cell and of determining differences in expression level above this threshold.
Highlights
Molecular imaging enables non-invasive characterisation of biochemical features at a molecular level, performed on anything from a living cell to an entire organism [1]
To determine the lower threshold of target abundance required for successful intranuclear imaging using RICs, we developed a model system expressing different levels of a well-characterised, stably expressed, nuclear localised target protein construct, histone 2B tagged enhanced green fluorescent protein (H2B-Enhanced Green Fluorescent Protein (EGFP))
To test the effects of target abundance on imaging ability, we generated a panel of H1299 cell lines that constitutively expressed Enhanced Green Fluorescent Protein (EGFP) tagged at its amino terminus with the histone protein H2B through stable transfection with the mammalian expression plasmid H2B-EGFP
Summary
Molecular imaging enables non-invasive characterisation of biochemical features at a molecular level, performed on anything from a living cell to an entire organism [1]. Whilst the vast majority of research has focused on the development of monoclonal antibodies targeting extracellular epitopes on either cancer cell membranes, the extracellular matrix, or epitopes shed into the interstitial space, approximately 30% of cellular proteins are localised within the nucleus, orchestrating a myriad of physiologically and pathologically relevant processes [3]. Antibodies are unable to cross cellular membranes due to their size (~150,000 Da) and hydrophilicity. These barriers can be overcome using cell penetrating peptides (CPPs) [4]. Cell penetrating peptides are short length peptides (less than 30 residues), that have the capacity to translocate across cellular membranes [5]. Many CPPs have been experimentally validated in vitro and in vivo to facilitate the translocation of bioactive molecular cargoes of various sizes, up to 540,000 kDa, across cellular membranes with limited toxicity [10]
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