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The influence of different methods of binding the OV-TL16 antibody and its Fab' fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer--drug (adriamycin [ADR] or meso chlorin e6 mono(N-2-aminoethylamide) (Mce6)) conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer--drug (ADR or Mce6) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling, the HPMA copolymer--Mce6 conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab' frgment of the OV-TL16 antibody demonstrated a more homogenous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physiocochemical methods were employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce6) and the spectral properties of Mce6 were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with the changes in antibody conformation.

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Our studies document a unique and unexpected advantage of the combination of HPMA copolymer bound doxorubicin with mesochlorin e6/photodynamic therapy in the treatment of ovarian cancer. Each drug's activity is individually enhanced when compared with free (low molecular weight) drugs, furthermore, in combination these HPMA copolymer bound agents act synergistically to create an unexpected biological effect. Figure 8 depicts the known activities of each agent which may play synergistic roles. HPMA copolymer-doxorubicin has been widely evaluated in preclinical and clinical studies. It demonstrates marked advantages over free doxorubicin: control of biodistribution and accumulation via molecular weight restrictions, biodegradability, minimal immunogenicity, subcellular localization, anticancer activity, enhanced permeability and retention, increased apoptosis, lipid peroxidation, DNA damage, and reduced nonspecific toxicity. Recent clinical trials in the UK provide "proof of principle" of the "enhanced permeability and retention effect" for solid tumors and the unique advantages of this novel drug delivery system for the treatment of ovarian cancer. With regards to photodynamic therapy using the photosensitizer mesochlorin e6, the preclinical evaluations thus far document: control of biodistribution and accumulation via molecular weight restrictions, biodegradability, subcellular localization, anticancer activity, enhanced permeability and retention, and reduced nonspecific toxicity. Ongoing microarray studies document unique cellular pathways and new pharmaceutical properties which are initiated by the HPMA copolymer delivery delivery of these agents, and predict an exciting future for this novel drug delivery system.