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Antibody-Indocyanin Conjugates for Immunophotodetection of Human Squamous Cell Carcinoma in Nude Mice¹

Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges [S. F., P. W., J-P. M.]; Institute of Environmental Engineering, Ecole Polytechnique Fédérale, CH-1015 Lausanne [D. B., G. W., H. v. d. B.] Switzerland; and Centre Val d'Aurelle, Service de Médecine Nucléaire, Rue de la Croix Verte, F-34094 Montpellier, France [A. P.]

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, a 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 µg of MAb E48-(indocyanin)₂ conjugate (representing 1 µg 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 µg of a MAb E48 coupled to 2 µg 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 µg) 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.

¹ This work was supported by the Swiss Foundation for Scientific Research, Grant 3229857.90.

² To whom requests for reprints should be addressed, at Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland.

Received 9/ 2/93. Accepted 3/21/94.

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