Folli S, Westermann P, Braichotte D, Pèlegrin A, Wagnières G, van den Bergh H, Mach J P
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Cancer Res. 1994 May 15;54(10):2643-9.
We have recently shown that immunophotodetection of human colon carcinomas in nude mice and in patients is possible by using anti-carcinoembryonic antigen monoclonal antibodies (MAb) coupled to fluorescein. The most common clinical application of photodiagnosis has been for the detection of squamous cell carcinomas (SCC) in the upper respiratory tract, but the free dyes used have a poor tumor selectivity. We selected the known MAb E48 directed against SCC and coupled it to a fluorescent dye: indopentamethinecyanin (indocyanin). This dye has an advantage over fluorescein in that it emits a more penetrating fluorescent red signal at 667 nm after excitation with a laser ray of 640 nm. In vitro, an conjugate with an indocyanin:MAb molar ratio of 2, and an additional trace labeling with 125I, showed more than 80% of binding to cells from the SCC line A431. In vivo, when injected i.v. into nude mice bearing xenografts of the same carcinoma line, the MAb E48-(indocyanin)2 conjugate was almost as efficient as the unconjugated MAb E48 in terms of specific tumor localization: 15% of the injected dose per g of tumor at 24 h after injection and a tumor:overall normal tissue ratio of 6-8. There was no selective tumor localization of an irrelevant IgG1-(indocyanin)2 conjugate. Immunophotodetection of the s.c. SCC xenografts on mice given injections of 100 micrograms of MAb E48-(indocyanin), conjugate (representing 1 microgram of indocyanin) was performed at 24 h. Upon laser irradiation, clearly detectable red fluorescence from the indocyanin-MAb conjugate was observed specifically in the SCC xenografts across the mouse skin. In comparison, injection of 100 micrograms of a MAb E48 coupled to 2 micrograms of fluorescein gave a specific green fluorescence signal in the tumor xenografts, which was detectable, however, only after removing the mouse skin. Injection i.v. of a 15 times higher amount of free indocyanin (15 micrograms) gave a diffuse red fluorescence signal all over the mouse body with no definite increase in intensity in the tumor, indicating a lack of tumor selectivity of the free dye. The results demonstrate the possibility of broadening and improving the efficiency of tumor immunophotodiagnosis by coupling to a MAb directed against SCC, a fluorescent dye absorbing and emitting at higher wavelength than fluorescein, and thus having deeper tissue penetration and lower tissue autofluorescence. Such a demonstration opens the way to a new form of clinical immunophotodiagnosis and possibly to the development of a more specific approach to phototherapy of early bronchial carcinomas.
我们最近发现，通过使用与荧光素偶联的抗癌胚抗原单克隆抗体（MAb），可以在裸鼠和患者体内对人结肠癌进行免疫光检测。光诊断最常见的临床应用是检测上呼吸道鳞状细胞癌（SCC），但使用的游离染料对肿瘤的选择性较差。我们选择了已知的针对SCC的MAb E48，并将其与一种荧光染料：吲哚五甲川菁（吲哚菁绿）偶联。这种染料比荧光素有一个优势，即在640nm激光激发后，它在667nm处发出穿透性更强的红色荧光信号。在体外，吲哚菁绿与MAb摩尔比为2的偶联物，以及用125I进行的额外微量标记，显示与SCC细胞系A431的细胞结合率超过80%。在体内，当静脉注射到携带相同癌细胞系异种移植物的裸鼠体内时，MAb E48-（吲哚菁绿）2偶联物在特异性肿瘤定位方面几乎与未偶联的MAb E48一样有效：注射后24小时，每克肿瘤摄取的注射剂量为15%，肿瘤与总体正常组织的比值为6 - 8。无关的IgG1-（吲哚菁绿）2偶联物没有选择性肿瘤定位。在给小鼠注射100微克MAb E48-（吲哚菁绿）偶联物（相当于1微克吲哚菁绿）后24小时，对皮下SCC异种移植物进行免疫光检测。激光照射后，在小鼠皮肤表面的SCC异种移植物中特异性地观察到来自吲哚菁绿-MAb偶联物的清晰可检测的红色荧光。相比之下，注射100微克与2微克荧光素偶联的MAb E48在肿瘤异种移植物中产生特异性绿色荧光信号，但只有在去除小鼠皮肤后才能检测到。静脉注射15倍量更高的游离吲哚菁绿（15微克）在小鼠全身产生弥漫性红色荧光信号，肿瘤部位的强度没有明显增加，表明游离染料缺乏肿瘤选择性。结果表明，通过将一种比荧光素吸收和发射波长更高、具有更深组织穿透性和更低组织自发荧光的荧光染料与针对SCC的MAb偶联，有可能拓宽和提高肿瘤免疫光诊断的效率。这样的证明为一种新的临床免疫光诊断形式开辟了道路，并可能为早期支气管癌的光治疗开发一种更具特异性的方法。
