Ilovich Ohad, Natarajan Arutselvan, Hori Sharon, Sathirachinda Ataya, Kimura Richard, Srinivasan Ananth, Gebauer Mathias, Kruip Jochen, Focken Ingo, Lange Christian, Carrez Chantal, Sassoon Ingrid, Blanc Veronique, Sarkar Susanta K, Gambhir Sanjiv S
From the Department of Radiology (O.I., A.N., S.H., A. Sathirachinda, R.K., A. Srinivasan, S.S.G.) and Departments of Bioengineering and Materials Science & Engineering (S.S.G.), Stanford University, 318 Campus Dr, Room E153, Stanford, CA 94305; Sanofi R&D, BioInnovation Novel Protein Therapeutics, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany (M.G., J.K., I.F., C.L.); Sanofi Oncology, Vitry, France (C.C., I.S., V.B.); and Sanofi Oncology, Cambridge, Mass (S.K.S.).
Radiology. 2015 Jul;276(1):191-8. doi: 10.1148/radiol.15140058. Epub 2015 Feb 27.
To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6.
Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests.
The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models.
Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.
开发并比较三种源自靶向CA6的抗体（huDS6）的铜64（64Cu）标记抗体片段，作为基于免疫正电子发射断层扫描（immuno-PET）的伴随诊断试剂，用于一种抗体药物偶联物，并使用huDS6进行研究。
制备、纯化了三种源自huDS6的抗体片段，将其与1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）偶联，并通过以下方式进行评估：（a）通过流式细胞术测量片段和DOTA偶联物的亲和力；（b）在人血清中离体测定标记片段在24小时内的稳定性；（c）通过连续PET成像和生物分布，评估片段在皮下接种CA6阳性和CA6阴性异种移植瘤的小鼠体内的成像潜力。使用抗溶菌酶和抗DM4 B-单链抗体片段的同型对照以及用过量的B-单链抗体片段或huDS6进行的阻断实验，以确定抗体片段64Cu-DOTA-B-单链抗体片段结合特异性的程度。分别通过细胞摄取和48小时成像实验评估免疫反应性和示踪动力学。使用t检验、单因素方差分析以及Wilcoxon和Mann-Whitney检验进行统计分析。
抗体片段64Cu-DOTA-B-单链抗体片段在人血清中24小时后稳定性超过95%，免疫反应性超过70%，并且在注射后6小时即可在体内区分CA6阳性和CA6阴性肿瘤，肿瘤之间的摄取率为1.7倍。同型和阻断研究实验表明，尽管在两种肿瘤模型中都有一些非特异性摄取，但示踪剂在抗原阳性肿瘤中有特异性摄取。
制备并检测了三种抗体片段作为潜在的伴随诊断试剂。64Cu-DOTA-B-单链抗体片段是一种用于体内CA6成像的稳定且有效的免疫PET示踪剂。
