Griffiths Gary L, Chang Chien-Hsing, McBride William J, Rossi Edmund A, Sheerin Agatha, Tejada German R, Karacay Habibe, Sharkey Robert M, Horak Ivan D, Hansen Hans J, Goldenberg David M
Immunomedics, Inc., Morris Plains, New Jersey. Garden State Cancer Center, Belleville, New Jersey 07950, USA.
J Nucl Med. 2004 Jan;45(1):30-9.
The aim of this work was to develop reagents and methods potentially useful in PET, using (68)Ga in a 2-step pretargeting protocol.
We prepared bispecific antibodies (bsAbs) for disease-specific targeting of carcinoembryonic antigen-positive cells and recognition of later-administered bivalent hapten-peptide conjugates. The secondary antibody arm (antibody 679) recognizes a histaminyl-succinyl-glycine (HSG) structural subunit. The bsAbs were prepared as Fab' x Fab' conjugates using chemical cross-linking methods and as bispecific diabodies using recombinant DNA technologies. A HSG-bivalent hapten conjugate bearing the macrocyclic ring chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) was designed to be readily radiolabeled with (68)Ga taken directly from a (68)Ge/(68)Ga generator system. Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer.
A chemically cross-linked hMN-14 x 679 F(ab')(2) and a fully humanized bispecific diabody construct (BS1.5H), expressed in Escherichia coli, were prepared for this work. We synthesized the bivalent peptide termed IMP 241 [DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH(2)] and labeled it with (68)Ga and (67)Ga at temperatures from 45 degrees C to 100 degrees C, over times of 15 min to 1 h, establishing 15 min at 95 degrees C as a useful condition for (68)Ga labeling. When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the (68)Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the (68)Ga into IMP 241, as analyzed by size-exclusion high-performance liquid chromatography, after mixing the complex with the 679 antibody. For in vivo studies we used (67)Ga-IMP 241 as a surrogate for (68)Ga-IMP 241, in view of the short, 68-min half-life of the (68)Ga nuclide. The (67)Ga-IMP 241 was successfully pretargeted to human colon tumor xenografts in athymic mice with both the chemical and the diabody bispecific proteins. High tumor-to-normal tissue ratios for (67)Ga uptake were found for all tissues at 1 to 6 h after injection of (67)Ga-IMP 241. When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of (67)Ga-IMP 241 at 1 and 3 h after injection were 41:1 and 137:1, 51:1 and 106:1, and 16:1 and 46:1, respectively.
The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of (68)Ga-labeled specific targeting agents in a routine clinical PET application.
本研究的目的是开发可能用于正电子发射断层扫描（PET）的试剂和方法，采用两步预定位方案使用（68）Ga。
我们制备了双特异性抗体（bsAbs），用于癌胚抗原阳性细胞的疾病特异性靶向以及识别随后给药的二价半抗原 - 肽缀合物。二抗臂（抗体679）识别组胺基 - 琥珀酰 - 甘氨酸（HSG）结构亚基。bsAbs采用化学交联方法制备为Fab'x Fab'缀合物，并采用重组DNA技术制备为双特异性双抗体。设计了一种带有大环环螯合剂1,4,7,10 - 四氮杂环十二烷 - N，N'，N“，N”'-四乙酸（DOTA）的HSG - 二价半抗原缀合物，以便直接用从（68）Ge/（68）Ga发生器系统获取的（68）Ga进行放射性标记。试剂先在体外进行测试，然后在人类癌症的临床前动物模型中测试其靶向特性。
为此项研究制备了一种化学交联的hMN - 14 x 679 F(ab')(2)和一种在大肠杆菌中表达的完全人源化双特异性双抗体构建体（BS1.5H）。我们合成了名为IMP 241的二价肽[DOTA - Phe - Lys(HSG) - D - Tyr - Lys(HSG) - NH(2)]，并在45℃至100℃的温度下，15分钟至1小时的时间内用（68）Ga和（67）Ga进行标记，确定95℃下15分钟是（68）Ga标记的有用条件。当我们用pH 4 - 5的乙酸铵缓冲液配制IMP 241二价半抗原 - 肽，并将发生器中的（68）Ga直接洗脱到肽溶液中时，在将复合物与679抗体混合后，通过尺寸排阻高效液相色谱分析，实现了（68）Ga几乎定量掺入IMP 241中。鉴于（68）Ga核素的半衰期短，仅68分钟，在体内研究中我们使用（67）Ga - IMP 241作为（68）Ga - IMP 241的替代物。（67）Ga - IMP 241通过化学和双抗体双特异性蛋白成功预定位到无胸腺小鼠的人结肠肿瘤异种移植瘤中。注射（67）Ga - IMP 241后1至6小时，所有组织中（67）Ga摄取的肿瘤与正常组织比率都很高。当使用BS1.5H双抗体进行预定位时，注射后1小时和3小时（67）Ga - IMP 241的肿瘤与血液、肿瘤与肝脏以及肿瘤与肺的比率分别为41:1和137:1、51:1和106:1以及16:1和46:1。
所描述的一般方法，以及我们开发的新组合物和标记方法，最终可能允许在常规临床PET应用中使用（68）Ga标记的特异性靶向剂。
