Rochefort Matthew M, Girgis Mark D, Knowles Scott M, Ankeny Jacob S, Salazar Felix, Wu Anna M, Tomlinson James S
Department of Surgery, UCLA, 570 Westwood Plaza, Bldg 114, CNSI, Rm4324E, Los Angeles, CA, 90095, USA,
Mol Imaging Biol. 2014 Oct;16(5):721-9. doi: 10.1007/s11307-014-0733-4.
Intact antibodies have a long serum persistence resulting in high background signal that inhibits their direct translation as imaging agents. Engineering of antibody fragments through the introduction of mutations in the fragment crystallizable (Fc) region can dramatically reduce serum persistence. We sought to develop a Fc-mutated, anti-CA19-9 antibody fragment (anti-CA 19-9 scFv-Fc H310A) to provide micro-positron emission tomography (microPET) imaging of pancreatic cancer xenografts.
The anti-CA19-9 scFv-Fc H310A was successfully expressed and purified. Biochemical characterization included size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and flow cytometry. The antibody fragment was labeled with iodine-124 ((124)I) and injected into mice containing human pancreatic cancer xenografts. MicroPET/CT images were then obtained. Blood, organ, and tumor radioactivity was measured and expressed as the percent of injected dose per gram of tissue (%ID/g).
Biochemical characterization was consistent with the creation of a 105 kD dimer containing a human Fc region. Flow cytometry demonstrated antigen-specific binding, and cell-based ELISA further established a dissociation constant (K D) of 10.7 nM. (124)I-labeled scFv-Fc H310A localized to the antigen-positive tumor xenografts as detected by microPET. Objective confirmation of targeting was demonstrated by higher %ID/g in the antigen-positive tumor compared to the blood, antigen-negative tumor, and liver.
We successfully engineered and produced an anti-CA19-9 scFv-Fc H310A antibody fragment that retains similar affinity when compared to the parental intact murine antibody. Additionally, our engineered and mutated fragment exhibited antigen-specific microPET imaging of both subcutaneous and orthotopic pancreatic cancer xenografts at early time points secondary to decreased serum half-life.
完整抗体在血清中的存留时间较长，会产生高背景信号，从而抑制其作为成像剂的直接应用。通过在可结晶片段（Fc）区域引入突变来构建抗体片段，可显著缩短其在血清中的存留时间。我们试图开发一种Fc区突变的抗CA19-9抗体片段（抗CA 19-9 scFv-Fc H310A），用于胰腺癌异种移植瘤的微型正电子发射断层扫描（microPET）成像。
成功表达并纯化了抗CA19-9 scFv-Fc H310A。生化特性鉴定包括尺寸排阻色谱、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质印迹法和流式细胞术。用碘-124（¹²⁴I）标记该抗体片段，并注射到携带人胰腺癌异种移植瘤的小鼠体内。然后获取microPET/CT图像。测量血液、器官和肿瘤的放射性，并表示为每克组织注射剂量的百分比（%ID/g）。
生化特性鉴定结果与含有人类Fc区的105 kD二聚体的构建一致。流式细胞术显示了抗原特异性结合，基于细胞的酶联免疫吸附测定（ELISA）进一步确定解离常数（KD）为10.7 nM。microPET检测显示，¹²⁴I标记的scFv-Fc H310A定位于抗原阳性的肿瘤异种移植瘤。与血液、抗原阴性肿瘤和肝脏相比，抗原阳性肿瘤中更高的%ID/g证实了靶向的客观存在。
我们成功构建并制备了抗CA19-9 scFv-Fc H310A抗体片段，与亲本完整鼠源抗体相比，其保留了相似的亲和力。此外，由于血清半衰期缩短，我们构建并突变的片段在早期对皮下和原位胰腺癌异种移植瘤均表现出抗原特异性microPET成像。
