Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.Bovine tuberculosis (bTB) continues to be a zoonotic disease affecting multiple species, including humans (3, 9, 21, 26). The disease has been difficult to control in livestock because of the lack of an effective vaccine, the presence of wildlife reservoirs, and the lack of a diagnostic assay with sufficient sensitivity and specificity to detect animals at all stages of infection. Currently, the primary methods used for the detection of TB in humans and ruminants include the measurement of a delayedtype hypersensitivity (skin test) to purified protein derivative (PPD) and an indirect in vitro assay that measures the concentration of gamma interferon (IFN-␥) produced in response to stimulation with PPD (22,30,31). Although the methods have proven useful in controlling bTB, they lack sensitivity and specificity because of a cross-reactive immune response to Tand B-cell epitopes conserved on orthologous molecules present in nonpathogenic mycobacteria and Mycobacterium avium subsp. paratuberculosis (reviewed in references 8, 23, and 27). To obviate this problem, an extensive effort has been under way to identify and characterize antigens unique to Mycobacterium bovis that could be used in a diagnostic assay. To date, studies have shown that the antibody response to M. bovis is not uniform, with no evidence of a dominant persistent response to a single antigen (reviewed in references 4, 7, and 8) at any stage of infection (2,19). This finding has suggested that some type of a multiplex assay is needed to detect animals at different stages of infection (1, 2). However, the necessity of using multiple antigens in an assay has introduced another challenge. The evaluation of the standard type of enzymelinked immunosorbent assay (ELISA) has shown that sensitivity and specificity are reduced when multiple antigens are combined for analysis in a single well, thus limiting the way a conventional ELISA can be used (20). To address this problem, we developed a multiplex assay that can simultaneously detect and analyze the response to multiple antigens spotted in a single well in a 96-well plate array format. We demonstrate the enhanced diagnostic power of a multiplex antigen approach over that of the industry-stand...

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L -like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L -like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L -like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L -like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis. [Mol Cancer Ther 2008;7(3):538 -47]

BackgroundProteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting.ResultsHere we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect.ConclusionsThis data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.

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The N-terminal propeptide domains of several cathepsin L-like cysteine proteases have been shown to possess potent inhibitory activity. Here we report the first kinetic characterisation of the inhibition properties of the cathepsin V propeptide (CatV PP). Using a facile recombinant approach we demonstrate expression, purification and evaluation of the CatV PP. This propeptide was found to behave as a tight-binding inhibitor against CatV (K (i) 10.2 nm). It also functions as an inhibitor against other members of the CatL-like subclass (CatL, 9.8 nm; CatS, 10.7 nm; and CatK, 149 nm) and had no discernible effects upon the more distantly related CatB.