A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.

A monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugate was prepared by a technique involving the site-specific covalent modification of the monoclonal antibody oligosaccharide moiety. Dextran carriers were synthesized with a single chain-terminal hydrazide group, which was used as the coupling point between the carrier and the monoclonal antibody carbohydrate. Selective in vitro photolysis of SK-MEL-2 human malignant melanoma cells was accomplished using several conjugates prepared from anti-melanoma 2.1 (chromophore:antibody molar ratios, 6.8 and 11.2). MATERIALS AND METHODSSynthesis of SnCe6-Dextran Carrier. The synthetic schemes used in the current work are outlined in Fig. 1 (17). Dextran (Pharmacia T-40; weight average molecular weight =35,000) was chosen as the carrier polymer because it is water soluble, is nontoxic, and possesses a single reducing terminus. A reactive hydrazide group was added to the reducing terminus via reductive amination with a large excess of apidic dihydrazide in the presence of sodium cyanoborohydride (18,19). Titration of the reducing end in I using the dinitrosalicylate method (20) showed a 92% modification of the terminal aldehyde relative to unaltered dextran. To prevent modification of the chain-terminal hydrazide in I prior to linkage to the antibody, a Trt protection group was introduced via the active ester TCPPH to yield II. In addition to providing a protected terminal hydrazide, the TCPPH was also designed to introduce a six-carbon spacer group on the dextran terminus in order to facilitate subsequent linkage to the mAb.

Monoclonal antibody (MAb)-dextran-tin(IV) chlorin e6 (SnCe6) immunoconjugates were prepared by a new technique involving the use of reducing, terminal-modified dextran carriers and site-specific modification of the Fc oligosaccharide moiety on the antibodies. Dextran carriers were synthesized to increase the number of SnCe6 molecules attached to a MAb. The dextran carriers were coupled to the MAb via a single, chain-terminal hydrazide group to prevent aggregation of MAbs. Conjugates were prepared with antimelanoma MAb 2.1 containing up to 18.9 SnCe6 molecules per MAb. Under neutral conditions, no hydrolysis of the hydrazone bond between the MAb and the dextran carrier could be detected, and the hydrazone was not stabilized by reduction with NaCNBH3 or NaBH4. Analysis of the purified immunoconjugates showed that approximately two dextran carrier chains were attached to a MAb regardless of the number of SnCe6 molecules linked to a dextran carrier. Site-specific covalent attachment of the SnCe6-dextran chains to the MAb was confirmed by SDS-PAGE. HPLC analysis of the conjugates gave a single species eluting in the range of 200-240 kDa. As determined by a competitive inhibition radioimmunoassay using viable SK-MEL-2 human malignant melanoma cells, the conjugates showed excellent retention of antigen-binding activity relative to unconjugated MAb.

Antibody-targeted photolysis is a technique for damaging or killing cells using light and an antibody-bound photosensitizer. In the present study, immunoconjugates were constructed to selectively kill Pseudomonas aeruginosa bacteria using tin (IV) chlorin e6 which was linked to dextran and then bound to the carbohydrate moiety of a monoclonal antibody specific for Pseudomonas aeruginosa Fisher type I polysaccharide antigen. Killing of Pseudomonas during mid-log phase growth was shown to be dependent upon light dose with complete bacterial cell killing observed at an irradiation dose of 80 J/cm2. Individual components of the immunoconjugates (e.g., antibody or chlorin alone) showed no bacterial cytotoxicity and immunoconjugates constructed with nonbinding antibodies were also ineffective as cytotoxic agents. These studies demonstrate that killing of gram negative bacteria using photoradiation is feasible and suggest that this methodology may be applicable in treatment of infections in man.