Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, Helmholtz Institute of RWTH Aachen University & Hospital, Pauwelsstraße 20, 52074 Aachen, Germany.
Cancer Immunol Immunother. 2012 Oct;61(10):1617-26. doi: 10.1007/s00262-012-1219-3. Epub 2012 Feb 19.
Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.
We transfected A-431 tumour cells with the far red-emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A' (ETA'). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.
The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA' resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.
We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA'.
治疗反应的临床前体内分析是评估新疗法的重要前提。远红（FR）/近红外（NIR）分子体内成像是一种很有前途的方法，因为它可以在单个动物的不同时间点进行测量，从而减少所需动物的数量，同时提高统计意义。在这里，我们展示了一种使用表达表皮生长因子受体（EGFR）的荧光细胞来监测治疗反应的方法，EGFR 是一种已经用于治疗的靶点。
我们用远红发射蛋白 Katushka（Kat2）转染 A-431 肿瘤细胞，当将其植入 BALB/c nu/nu 小鼠中并用 CRi Maestro 活体成像仪进行监测时，会产生强烈的荧光，从而可以监测肿瘤的生长。我们用以前报道的免疫毒素（IT）靶向 A-431 细胞，该免疫毒素由抗 EGFR 抗体单链可变片段（scFv）425 与铜绿假单胞菌外毒素 A'（ETA'）融合而成。此外，EGFR 的表达也通过将与 NIR 染料 BG-747 偶联的 425(scFv)通过 SNAP 标签连接子进行验证。
结果表明，通过 FR 成像评估体内治疗反应，并在同一位置检测 EGFR 表达是可行的。用 425(scFv)-ETA'治疗会导致肿瘤生长速度减慢，而不会影响动物的整体健康状况。这与多柔比星的治疗形成对比，多柔比星虽然能减小肿瘤体积，但会导致健康状况不佳。
我们开发了一种新的方法，通过活体成像对多个参数进行非侵入性地确定治疗反应，该方法显示了 425(scFv)-ETA'的疗效。
