Design and synthesis of MMP inhibitors with appended fluorescent tags for imaging and visualization of matrix metalloproteinase enzymes.

We describe the synthesis, MMP-2 and 9 potency, and in vitro evaluation of a series of α-sulfone hydroxmate MMP inhibitors conjugated to a series of dyes with different absorption/emission lamina maxima's that can be used to visualize tumors.

Non-destructive and selective imaging of the functionally active, pro-invasive membrane type-1 matrix metalloproteinase (MT1-MMP) enzyme in cancer cells.

Proteolytic activity of cell surface-associated MT1-matrix metalloproteinase (MMP) (MMP-14) is directly related to cell migration, invasion, and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR, and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma, and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1 × 10(5) molecules/cell, whereas these levels are in a 1 × 10(6) range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.

Antibody targeting of cell-bound MUC1 SEA domain kills tumor cells.

The cell-surface glycoprotein MUC1 is a particularly appealing target for antibody targeting, being selectively overexpressed in many types of cancers and a high proportion of cancer stem-like cells. However the occurrence of MUC1 cleavage, which leads to the release of the extracellular α subunit into the circulation where it can sequester many anti-MUC1 antibodies, renders the target problematic to some degree. To address this issue, we generated a set of unique MUC1 monoclonal antibodies that target a region termed the SEA domain that remains tethered to the cell surface after MUC1 cleavage. In breast cancer cell populations, these antibodies bound the cancer cells with high picomolar affinity. Starting with a partially humanized antibody, DMB5F3, we created a recombinant chimeric antibody that bound a panel of MUC1+ cancer cells with higher affinities relative to cetuximab (anti-EGFR1) or tratuzumab (anti-erbB2) control antibodies. DMB5F3 internalization from the cell surface occurred in an efficient temperature-dependent manner. Linkage to toxin rendered these DMB5F3 antibodies to be cytotoxic against MUC1+ cancer cells at low picomolar concentrations. Our findings show that high-affinity antibodies to cell-bound MUC1 SEA domain exert specific cytotoxicity against cancer cells, and they point to the SEA domain as a potential immunogen to generate MUC1 vaccines.