Diagnostic imaging requires the sufficient intensity of a corresponding signal from an area of interest in order to obtain diagnostically significant images. The contrast agents are specific for each imaging modality, and the tissue concentration required for successful imaging varies between diagnostic moieties in broad limits. Liposomes and micelles have been proposed as in vivo carriers for various diagnostic agents in order to increase their local concentration. The use of antibody-mediated targeted delivery of diagnostic agents to the areas of interest (tumors) was also proposed. To attach metals serving as radioisotopes and paramagnetic moieties to an antibody, chelating groups are introduced into a protein molecule. To couple many reporter groups to a single antibody molecule, the single-point modification of an antibody with polymers carrying multiple chelating residues (polychelating polymers, PPs) was proposed providing the major increase in the amount of heavy metal binding sites per antibody molecule without affecting the antibody properties. Polychelating amphiphilic polymers (PAPs) prepared by modifying PPs with a hydrophobic fragment capable of incorporation into the liposome membrane or the micelle core, have been used for liposome and micelle loading with diagnostic metals. Micelle-forming iodine-containing polymers have also been suggested as the contrast agents for computed tomography (CT). The attachment of an antibody to contrast-loaded polymers, liposomes, and micelles should enhance contrast accumulation in the targets; however, diagnostic monoclonal antibodies are usually tumor type-specific and unable to react with a broad variety of tumors. In recent years, we have identified a subset of natural antibodies capable of binding to the surface of a variety of cancer cells but not normal cells. These antibodies belong to natural anti-nuclear auto-antibodies (ANAs), which can be found in healthy mammals, possess the nucleosome (NS)-restricted specificity, and recognize the surface of numerous tumor cells both in vitro and in vivo via tumor cell surface-bound NSs as was clearly shown for two monoclonal tumor-specific ANAs (TSANAs), 2C5 and 1G3. We hypothesize that the combination of these two approaches - the attachment of antibodies or their fragments with broad anti-cancer specificity to contrast-loaded polychelating polymers and liposomes and micelles containing such polymers - will bring to life a new generation of highly efficient targeted imaging agents for cancer suitable for various imaging modalities depending on the type of a contrast moiety used. The proposed research pursues the following four specific aims to test our hypothesis: 1. To purify 2C5 and 1G3 and their Fab fragments; attach them to pre-synthesized PPs and PAP-containing liposomes and micelles; and investigate the immunoreactivity of these conjugates and their ability to bind cancer cells (murine EL4 T lymphoma, Lewis lung carcinoma, or B16.F10 melanoma and human BT20 and MCF7 breast adenocarcinomas) in vitro. 2. To investigate the biodistribution and tumor accumulation (tumor-to normal ratio) of the preparations demonstrating a good combination of loading capacity for different reporter groups and ability to specifically bind cancer cells in vitro in normal and nude mice bearing the tumors listed above. 3. To perform the gamma-imaging of the listed murine and human tumor in vivo in mice with 111-1n-loaded 2C5(or 1G3)-containing nanoparticular polymeric contrasts. 4. To perform the MR imaging of the listed murine and human tumors in mice with Gd-loaded PAP-containing immunoliposomes or immunomicelles and their CT imaging with the use of antibody- or Fab-modified iodine-containing micelles.
