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.

鉴于其多功能性、特异性和敏感性，使用标记到细胞穿透肽的抗体对核内表位进行成像具有巨大的潜力。然而，由于目标的位置，这个过程在技术上具有挑战性。先前的研究已经证明了可以用基于抗体的放射免疫偶联物靶向的多种核内表位。在这里，我们开发了一种核定位绿色荧光蛋白 (GFP) 的受控表达模型，以使用放射免疫偶联物询问核内 SPECT 的技术限制，特别是较低的目标丰度检测阈值。方法：我们用增强的 GFP (EGFP) 标记的组蛋白 2B (H2B) 稳定转染肺腺癌细胞系 H1299，并产生了 4 个表达增加水平的 GFP 的细胞系。使用蛋白质印迹、流式细胞术和酶联免疫吸附测定法对 EGFP 水平进行量化。抗 GFP 抗体 (GFP-G1) 使用基于二苯并环辛炔-N ₃的菌株促进的叠氮化物-炔烃环加成与细胞穿透肽 TAT (GRKKRRQRRRPPQGYG) 进行修饰，其中还包括核定位序列和金属离子螯合剂N ₃ -Bn-二乙烯三胺五乙酸酯 (DTPA) 允许用¹¹¹ In 进行放射性标记。细胞摄取¹¹¹In-GFP-G1-TAT 在体外表达不同水平的 H2B-EGFP 的 5 个细胞克隆进行了评估。^{对异种移植小鼠的肿瘤摄取进行量化，以确定使用111} In-GFP-G1-TAT可检测到的最小靶表位量。结果：我们生成了 4 个 H1299 细胞克隆，表达不同水平的 H2B-EGFP（每个细胞 0-1 百万个拷贝，包括野生型 H1299 细胞）。GFP-G1 单克隆抗体在内部生产和纯化，并证实了与 H2B-EGFP 的选择性结合。GFP-G1 的亲和力（解离常数）确定为 9.1 ± 3.0 nM。GFP-G1 与 TAT 和 DTPA 结合。¹¹¹表达 H2B-EGFP 的细胞克隆中的 In-GFP-G1-TAT 摄取与 H2B-EGFP 表达呈线性相关（P< 0.001)。体内异种移植研究表明，肿瘤组织中的¹¹¹ In-GFP-G1-TAT 摄取与 H2B-EGFP 的表达呈线性相关（P = 0.004），并表明每个细胞约 240,000 拷贝的较低目标丰度检测阈值。结论：在这里，我们提出了一个概念验证证明，即基于抗体的核内靶标成像能够检测每个细胞拷贝数高于 240,000 拷贝的感兴趣表位的存在，并确定上述表达水平的差异这个门槛。