The development and characterisation of porphyrin isothiocyanate-monoclonal antibody conjugates for photoimmunotherapy.

A promising approach to increase the specificity of photosensitizers used in photodynamic therapy has been through conjugation to monoclonal antibodies (MAb) directed against tumour-associated antigens. Many of the conjugations performed to date have relied on the activated ester method, which can lead to impure conjugate preparations and antibody crosslinking. Here, we report the development of photosensitizer-MAb conjugates utilising two porphyrin isothiocyanates. The presence of a single reactive isothiocyanate allowed facile conjugation to MAb FSP 77 and 17.1A directed against internalizing antigens, and MAb 35A7 that binds to a non-internalizing antigen. The photosensitizer-MAb conjugates substituted with 1-3 mol of photosensitizer were characterised in vitro. No appreciable loss of immunoreactivity was observed and binding specificity was comparable to that of the unconjugated MAb. Substitution with photosensitizer had a minimal effect on antibody biodistribution in vivo for the majority of the conjugates, although a decreased serum half-life was observed using a cationic photosensitizer at the higher loading ratios. Tumour-to-normal tissue ratios as high as 33.5 were observed using MAb 35A7 conjugates. The internalizing conjugate showed a higher level of phototoxicity as compared with the non-internalizing reagent, using a cell line engineered to express both target antigens. These data demonstrate the applicability of the isothiocyanate group for the development of high-quality conjugates, and the use of internalizing MAb to significantly increase the photodynamic efficiency of conjugates during photoimmunotherapy.

Internalisation enhances photo-induced cytotoxicity of monoclonal antibody-phthalocyanine conjugates.

Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of the conjugated dye. To analyze the potential role of internalisation of the dye on photo-induced cytotoxicity, we compared two target antigens, carcinoembryonic antigen (CEA) that does not internalise and ErbB2 that does. Human ovarian carcinoma SKOv3 cells that express a high level of ErbB2 were transfected with the CEA cDNA. Using FACS analysis, the resulting cell line, SKOv3-CEA-1B9, demonstrated comparable levels of expression of the two target antigens. Aluminium tetrasulfophthalocyanine (AlPcS(4)) was covalently coupled to anti-CEA MAb 35A7, anti-ErbB2 MAb FSP77 and a non-specific MAb PX, via a five-carbon sulfonamide spacer chain (A(1)) at molar ratios ranging from 6 to 9 moles of AlPcS(4) per mole of MAb. The 35A7-(AlPcS(4)A(1))(8) conjugate induced 68% growth inhibition of the SKOv3-CEA-1B9 cell line after a 20 h incubation at 2.50 microg/ml (based on AlPcS(4)A(1) content) following light exposure. However, the FSP77-(AlPcS(4)A(1))(6) conjugate gave a 51% growth inhibition for an AlPcS(4)A(1) concentration as low as 0.04 microg/ml after the same incubation time and exposure to the same light dose. At a 1.25 microg/ml AlPcS(4)A(1) concentration, the FSP77-(AlPcS(4)A(1))(6) conjugate gave a 67% growth inhibition after an incubation time as short as 1 h, reaching a 96% inhibition after an 8 h incubation time. Using an unique cell line that expresses two different target antigens, we demonstrated a clear advantage of an internalising over a non-internalising MAb-dye conjugate in terms of phototoxic efficacy. In vivo evaluation of the photodynamic properties of the conjugates is in progress.

Intraoperative immunophotodetection for radical resection of cancers: evaluation in an experimental model.

The aim of our study was to assess the technique of immunophotodetection (IPD) in intraoperative situations in an experimental model and to determine its capacity to detect very small tumor masses. IPD is a recent technology involving fluorescent dye-labeled monoclonal antibodies (MAbs) directed against tumor-associated antigens. Up to now, no intraoperative device for IPD has been developed, and limits of detection of the technique are unknown. MAb-dye conjugates were prepared using the anti-carcinoembryonic antigen MAb 35A7 labeled with indocyanine and (125)I. Time-dependent (6, 12, 24, 48, and 96 h post i.v. injection) and dose-dependent (10, 40, and 100 microg of conjugate) biodistribution studies were performed in nude mice bearing an LS174T peritoneal carcinomatosis demonstrating high tumor uptake (up to 21% of the injected dose/g of tumor 48 h postinjection). Intraoperative IPD was studied, using a newly developed device, in 16 mice 48 h after i.v. injection of 40 microg of the (125)I-MAb 35A7-indocyanine conjugate. The fluorescent status of 333 biopsies was compared with their histological analysis. Sensitivity was 90.7%, specificity was 97.2%, the positive predictive value was 94.7%, and the negative predictive value was 94.9%. Detection of very small nodules (<1 mg in weight or <1 mm in diameter) was possible. However, we observed a decrease in sensitivity as a function of tumor mass: 100% for nodules >10 mg versus 78% for nodules < or =1 mg. These experiments demonstrate that intraoperative IPD is easy to use and associated with high sensitivity and specificity, even for low tumor masses. On the basis of these encouraging results, intraoperative IPD should be assessed in a clinical study.

[Cancer immunophotodetection and immunophototherapy]. / Immunophotodétection et immunophotothérapie des cancers.

Some dyes can be used for cancer diagnosis and therapy. Following administration, the dye concentrate with some specificity in the tumor and can react to an irradiation with light. Some excited dyes can be fluorescent allowing detection of very small tumors. Some other dye can be phototoxic and lead to the destruction of the tumor. These attractive techniques are however limited because of the too low tumor specificity of the currently used dyes (hematoporphyrin and phthalocyanine). A new strategy has be developed in which the fluorescent or phototoxic dye is coupled to an antibody directed against a tumor associated antigen. This approach allow the selection of the dye only for its optimal photochemical properties, the antibody being used as carrier to concentrate the dye in the tumor. Many experimental immunophototherapy studies have been performed with different phototoxic dyes. Some results are encouraging but the involved mechanisms are complex and they limit the current clinical applications of immunophototherapy. Concerning immunophotodetection, two dyes have been coupled to anti-tumor antibodies: fluorescein and indocyanin. The antibody-dye conjugates have been evaluated in experimental studies and in pilot clinical trials. The most recent results concern the use of this technique in intraoperative situation and to visualize neo-vascularization. Immunophotodetection gives a very precise image of tumors. The detected tumor nodules are in the millimeter range. However, according to the limited light pathway in the tumor, this technique can be applied only to cutaneous areas, endoscopy accessible areas and intraopearative situations. Immunophotodetection is an attractive imaging technique using antibodies.

Preparation, phototoxicity and biodistribution studies of anti-carcinoembryonic antigen monoclonal antibody-phthalocyanine conjugates.

Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of a conjugated dye. Aluminum tetrasulfophthalocyanine (AlPcS4) was covalently coupled to a 35A7 MAb directed against carcinoembryonic antigen (CEA) via a five-carbon spacer chain (A1) to yield conjugates with a molar ratio ranging from 5 to 16 mol of AlPcS4 per mol of 35A7 MAb. Conjugates were labeled with radioiodine for characterization. The immunoreactivity of the conjugates, determined in a direct binding assay on CEA coupled to sepharose, was not modified by the coupled AlPcS4A1 molecules. In vivo, these conjugates were evaluated in nude mice bearing human colon carcinoma xenografts (T380). 35A7 MAb-(AlPcS4A1)5, 35A7 MAb-(AlPcS4A1)12 and 35A7 MAb-(AlPcS4A1)16 conjugates displayed a tumor uptake of 35 +/- 5.0%, 40 +/- 5.7% and 32 +/- 3.3% of the injected dose per gram of tumor tissue, respectively, corresponding to an uptake of 97%, 104% and 91% as compared to that of the unconjugated 35A7 MAb. In each experimental group, the tumor-to-normal tissue ratios obtained with the conjugates were almost identical to those obtained with unconjugated 35A7 MAb. Average values of 1.8, 7 and about 30 were obtained for blood, liver and muscle, respectively. Phototoxic efficacy of the 35A7 MAb-(AlPcS4A1)12 conjugate was demonstrated in vitro on the LoVo cell line giving a 91% growth inhibition for a 2.50 micrograms/mL AlPcS4A1 concentration. We conclude that these conjugates demonstrate clear in vivo tumor-seeking capacity and in vitro photocytotoxic properties. Such conjugates could thus be promising candidate drugs for clinical photodynamic therapy of cancers expressing CEA.