Silvio Folli

Antibody–fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice

To improve the detectability of tumors by light‐induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein‐MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein–MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I‐MoAb–(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti‐CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 μg of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 μg of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser‐induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

Long circulating half-life and high tumor selectivity of the photosensitizer meta-tetrahydroxyphenylchlorin conjugated to polyethylene glycol in nude mice grafted with a human colon carcinoma

In a model of nude mice bearing a human colon carcinoma xenograft, the biodistribution and tumor localization of meta‐tetrahydroxyphenylchlorin (m‐THPC) coupled to polyethylene glycol (PEG) were compared with those of the free form of this photosensitizer used in photodynamic therapy (PDT). At different times after i.v. injection of both forms of 125I‐labeled photosensitizer, m‐THPC‐PEG gave on average a 2‐fold higher tumor uptake than free m‐THPC. In addition, at early times after injection, m‐THPC‐PEG showed a 2‐fold longer blood circulating half‐life and a 4‐fold lower liver uptake than free m‐THPC. The tumor to normal tissue ratios of radioactivity concentrations were always higher for m‐THPC‐PEG than for free m‐THPC at any time point studied from 2 to 96 hr post‐injection. Significant coefficients of correlation between direct fluorescence measurements and radioactivity counting were obtained within each organ tested. Fluorescence microscopy studies showed that m‐THPC‐PEG was preferentially localized near the tumor vessels, whereas m‐THPC was more diffusely distributed inside the tumor tissue. To verify whether m‐THPC‐PEG conjugate remained phototoxic in vivo, PDT experiments were performed 72 hr after injection and showed that m‐THPC‐PEG was as potent as free m‐THPC in the induction of tumor regression provided that the irradiation dose for m‐THPC‐PEG conjugate was adapted to a well‐tolerated 2‐fold higher level. The overall results demonstrate first the possibility of improving the in vivo tumor localization of a hydrophobic dye used for PDT by coupling it to PEG and second that a photosensitizer conjugated to a macromolecule can remain phototoxic in vivo. Int. J. Cancer 76:842–850, 1998.© 1998 Wiley‐Liss, Inc.

Photodetection of early cancer in the upper aerodigestive tract and the bronchi using photofrin II and colorectal adenocarcinoma with fluoresceinated monoclonal antibodies

The performance of a fluorescence endoscope for the detection of early cancer is clinically evaluated. the apparatus is based on the imaging of the laser-induced fluorescence (LIF) of a dye which localizes in the tumor after IV injection with a higher concentration than in the surrounding normal tissue. The tests are carried out in several of the hollow organs, such as the upper aerodigestive tract, the bronchi, and the colon. In the two former cases the dye used is photofrin II, whereas in the latter case conjugates between monoclonal antibodies (Mab) directed against carcinoembrionic antigen (CEA) and fluorescein molecules are injected. The fluorescence contrast between tumor and surrounding tissue is enhanced by real-time image processing which eliminates most of the tissue autofluorescence as well as the fluorescence due to the relatively small amount of dye localized in the normal tissue. This is done by recording the fluorescence image in two spectral domains, after which these two images are digitized and manipulated with a mathematical operator (lookup-table). The sources of false positives and false negatives are evaluated in terms of the fluorescent dye and tissue optical properties.

Carcinoembryonic antigen expression, antibody localisation and immunophotodetection of human colon cancer liver metastases in nude mice : A model for radioimmunotherapy

Colorectal cancer frequently disseminates through the portal vein into the liver. In this study, outbred Swiss nude mice were adapted to facilitate the induction of liver metastases by a pre‐grafting treatment with 6 Gy total body irradiation and i.v. injection of anti‐asialo GMI antibody. One day later, cultured LS 174T human colon cancer cells were injected into the surgically exposed spleen, which was resected 3 min later. In 48 of 65 mice, a few to several hundred liver metastases were macroscopically observed at dissection 3 to 4 weeks after transplantation. Ten of 10 mice, followed‐up for survival, died with multiple large confluent liver metastases. By reducing the radiation dose to 4 or 0 Gy, or omitting the anti‐asialo GMI antibody injection, only 60%, 37% or 50% of mice, respectively, had visible metastases 3 weeks after transplantation. Carcinoembryonic antigen (CEA) measured in tumour extracts was in the mean 25.6 μg/g in liver metastases compared with 9.2 μg/g in s.c. tumours. Uptake of radiolabelled anti‐CEA monoclonal antibody (MAb) in the metastases 12, 24 and 48 hr after injection gave a mean value of 39% of the injected dose per gram of tissue (ID/g). In comparison, MAb uptake in s.c. and intrasplenic tumours or lung metastases gave a mean percentage ID/g of 20, 18 and 15, respectively. Laser‐induced fluorescence after injection of Indocyanin‐MAb conjugate allowed direct visual detection of small liver metastases, including some that were not visible under normal light. Preliminary results showed that mice, pre‐treated with 4 Gy irradiation and the anti‐asialo GMI injection, were tolerant to radioimmunotherapy with a total dose of 500 μCi 131I labeled anti‐CEA intact MAbs given in 3 injections.

Higher Efficiency of 131I-Labeled Anti-Carcinoembryonic Antigen—Monoclonal Antibody F(ab′)2 as Compared to Intact Antibodies in Radioimmunotherapy of Established Human Colon Carcinoma Grafted in Nude Mice

Radioimmunotherapy using 131I-labeled antibodies has been shown to be very efficient in patients with B cell lymphoma: 85% of patients responded with complete remission upon myeloablative therapy using radiolabeled MB-1 and B1 antibody (Press et al. 1993). In treatment of solid tumors, however, such response rates have not been reached (Mach et al. 1991; Goldenberg and Schlom 1993; Breitz et al. 1992).

Comparison of the influence of a water-soluble polymer carrier on the tumor localization and biodistribution of mesotetrametahydroxyphenylchlorin (mTHPC) in two animal models

Mesotetrametahydroxyphenylchlorin (mTHPC) is compared to mTHPC derivatized with one or more polyethyleneglycol (PEG) chains of about 6000 amu. The latter compound is called mTHPC-PEG hereafter. In particular the uptake and removal of these two compounds is studied in tumor and other tissues after injection as a function of the time. In a first model this is done with nude mice bearing human colorectal xenografts (LS174T); in a second model it is studied in a Chinese hamster with a 7,12 dimethylbenzanthracene (DMBA) induced early squamous cell carcinoma in the cheekpouch mucosa. In the nude mice with tumors grown to between 70 and 130 mg, tumor to muscle dye ratios are found to attain a maximum value of 4.8, eight hours after injection of mTHPC. With mTHPC-PEG the tumor to muscle ratio attained a value as high as 18.6, forty-eight hours after injection. In the hamster model the maximum tumor to normal mucosa ratio for mTHPC was about 3.5 at times up to 2 hours after injection. For the mTHPC-PEG this ratio is always near unity in this model.

Immunophotodetection of Cancer by Antibody-Indocyanin Conjugates

We have recently shown that immunophotodetection of human colon carcinomas in nude mice and in patients is possible by using anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) coupled to fluorescein. Here we show, by using a new fluorescent dye, indocyanin, coupled to either anti-CEA or anti-squamous cell carcinoma MAbs, that the efficiency of tumor immunophotodetection in nude mice bearing either human colon carcinoma or squamous cell carcinoma xenografts, can be markedly improved. This improvement is due to the higher absorption and emission wavelengths of indocyanin (652 – 667 nm) as compared to fluorescein (488–515 nm), which give a deeper tissue penetration and lower tissue autofluorescence. Preliminary clinical studies in patients with SCC, however, showed that the tumor to normal tissue ratios obtained after injection of 125I-MAb E48-indocyanin conjugates were not sufficient to allow satisfactory immunophotodiagnosis. In contrast, when we used our chimeric anti-CEA antibody coupled to indocyanin in patients with colon carcinomas, we obtained very elegant and specific tumor immunophotodetection. The overall results demonstrate the feasibility of a new form of in vivo tumor diagnosis called immunophotodetection, which may open the way to the development of a more specific approach to phototherapy of early carcinomas.