Freise Amanda C, Zettlitz Kirstin A, Salazar Felix B, Lu Xiang, Tavaré Richard, Wu Anna M
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Crump Institute for Molecular Imaging, University of California, 570 Westwood Plaza, CNSI, PO Box 951770, Los Angeles, CA, 90095-1770, USA.
Department of Internal Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Mol Imaging Biol. 2017 Aug;19(4):599-609. doi: 10.1007/s11307-016-1032-z.
PURPOSE: Molecular imaging of CD4 T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4 T cell viability, proliferation, CD4 expression, and function. PROCEDURES: The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed. RESULTS: For immunoPET imaging, the lowest protein dose of 2 μg of Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-μg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 μg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 μg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 μg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4 T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4 T cells. CONCLUSIONS: Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4 T cells in the context of preclinical disease models. Future approaches to minimizing biological effects may include the creation of monovalent fragments or selecting anti-CD4 antibodies which target alternative epitopes.
目的：对全身CD4 T细胞进行分子成像有助于监测自身免疫性疾病和癌症免疫治疗。鉴于这些细胞在调节免疫方面的关键作用，开发一种生物惰性探针很重要。先前开发的抗小鼠CD4抗体片段GK1.5半胱氨酸双体（cDb）在不同剂量下进行测试，以评估其对正电子发射断层扫描（PET）成像以及CD4 T细胞活力、增殖、CD4表达和功能的影响。 程序：通过给小鼠注射不同量的锆标记GK1.5 cDb，随后进行PET成像和离体生物分布分析，评估蛋白质剂量对图像对比度（淋巴组织与肌肉比值）的影响。为评估GK1.5 cDb对T细胞生物学的影响，将GK1.5 cDb与T细胞在体外孵育，或以多种蛋白质剂量静脉注射到C57BL/6小鼠体内。用流式细胞术分析CD4表达和T细胞增殖，并检测细胞因子。 结果：对于免疫PET成像，与12μg蛋白质剂量相比，最低蛋白质剂量2μg的锆标记GK1.5 cDb导致腹股沟淋巴结（ILN）和脾脏中每克注射剂量百分比显著更高。以40μg的高剂量在体内给予GK1.5 cDb导致脾脏、血液、淋巴结和胸腺中CD4表达短暂下降，在注射后3天内恢复；当给予2μg时，这种效应有所降低，尽管未消除。通过注射40μg GK1.5 cDb，ILN中的增殖在体内受到抑制，但脾脏中未受抑制。体外，超过25nM的GK1.5 cDb浓度显著抑制CD4 T细胞增殖和干扰素-γ产生。总体而言，使用低剂量GK1.5 cDb可将对CD4 T细胞的生物学影响降至最低。 结论：低剂量GK1.5 cDb可产生高对比度免疫PET图像，对体外和体内T细胞生物学影响最小，可能是在临床前疾病模型中研究CD4 T细胞的有用工具。未来将生物学影响降至最低的方法可能包括创建单价片段或选择靶向替代表位的抗CD4抗体。
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