RT Journal Article
SR Electronic
T1 A Model System to Explore the Detection Limits of Antibody-Based Immuno-SPECT Imaging of Exclusively Intranuclear Epitopes
JF Journal of Nuclear Medicine
JO J Nucl Med
FD Society of Nuclear Medicine
SP 1537
OP 1544
DO 10.2967/jnumed.120.251173
VO 62
IS 11
A1 Mathew Veal
A1 Gemma Dias
A1 Veerle Kersemans
A1 Deborah Sneddon
A1 Stephen Faulkner
A1 Bart Cornelissen
YR 2021
UL http://jnm.snmjournals.org/content/62/11/1537.abstract
AB 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 in vitro. 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.