A Model System to Explore the Detection Limits of Antibody-Based Immuno-SPECT Imaging of Exclusively Intranuclear Epitopes.
J Nucl Med
; 62(11): 1537-1544, 2021 11.
Article
em En
| MEDLINE
| ID: mdl-33789931
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 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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Tomografia Computadorizada de Emissão de Fóton Único
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article