AIMS/HYPOTHESIS Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody '4hD29') against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography (PET). METHODS DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-βH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-βH1 cells (2 × 106, 5 × 106 and 10 × 106 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using 99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N-chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either 67Ga (SPECT) or 68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. RESULTS The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-βH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-βH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. CONCLUSIONS/INTERPRETATION These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes.

中文翻译：

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靶向二肽基肽酶6的基于纳米抗体的核成像示踪剂，以确定小鼠中人β细胞移植物的质量。

目的/假设1型糖尿病的特征在于β细胞量的逐渐减少。胰岛同种异体移植后也观察到了这一点，但没有关于β细胞丢失的时间过程的可靠信息。这是由于非侵入性胰岛成像技术的可用性有限。我们以前曾描述过，二肽基肽酶6（DPP6）是一种α和β细胞特异性生物标记，并开发了针对它的骆驼科动物抗体（nanobody'4hD29'）。我们证明了通过单光子发射计算机断层扫描（SPECT）/计算机断层扫描（CT）检测表达DPP6的细胞的可能性，但未评估移植的细胞数量与SPECT信号之间的相关性。这里，我们调查4hD29纳米抗体是否允许我们检测植入免疫缺陷小鼠的不同量的人类胰岛。此外，我们还描述了该探头与正电子发射断层扫描（PET）配合使用的适应性。方法使用组织芯片和免疫组化技术评估人样本中DPP6的表达。使用Hoechst /碘化丙啶染色和抗胰岛素ELISA分别测量了EndoC-βH1细胞和人胰岛中4hD29纳米抗体对细胞死亡和葡萄糖刺激的胰岛素分泌的影响。我们对使用不同量的EndoC-βH1细胞（2×106、5×106和10×106细胞），人胰岛（1000和3000）或胰腺外分泌组织移植的严重联合免疫缺陷（SCID）小鼠进行了体内SPECT成像99mTc标记的4hD29纳米抗体。该DPP6纳米抗体还与N-氯代琥珀酰亚胺（NCS）-1,4,7-三氮杂环壬烷-1,4,7-三乙酸（NOTA）偶联，用67Ga（SPECT）或68Ga（PET）进行放射性标记，并用于原理验证实验，用于检测SCID小鼠中移植的DPP6表达细胞（凯利神经母细胞瘤）。结果DPP6蛋白主要在胰岛中表达。重要的是，抗DPP6纳米抗体4hD29可以无创地检测出免疫缺陷小鼠中移植的大量EndoC-βH1细胞或人类胰岛。这表明必须进一步改进探针以检测更少数量的胰岛细胞。4hD29纳米抗体既不影响β细胞活力，也不改变EndoC-βH1细胞和人类胰岛中的胰岛素分泌。4hD29纳米抗体成功转化为PET探针，并且没有改变其特异性。