Antibody targeting of Cathepsin S induces antibody-dependent cellular cytotoxicity.

BACKGROUND: Proteolytic 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. RESULTS: Here 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. CONCLUSIONS: This 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.

Expression and purification of diagnostically sensitive mycobacterial (Mycobacterium bovis) antigens and profiling of their humoral immune response in a rabbit model.

The incidence of bovine tuberculosis (bTB) is increasingly giving rise to large economic losses in the agricultural industry. The current methods used for detection and control of bTB (skin test and interferon-gamma) lack desired sensitivity and specificity. Therefore, the development of a rapid and reliable bTB serological based assay is urgently required. An antibody assay using combinations of strain-specific mycobacterial antigens could resolve both specificity and sensitivity issues. We analyzed the ability of a series of selected mycobacterial antigens to outline a humoral immune response in a rabbit model experimentally challenged with different mycobacterium. Antibodies specific for three antigens, MTB40, ESAT6 and CFP10, were present in serum 2 weeks post-challenge (early indicator), while two other antigens, Rv3870 and Rv1580c, could be detected from 8 to 11 weeks post-challenge. These selected mycobacterial antigens did not exhibit any cross-reactivity with avian PPD and only a very low positivity with bovine PPD. This data suggests that this panel of strain-specific mycobacterial antigens could be used for identification of Mycobacteriumbovis infection in serum samples. The combinatorial application of these antigens could form part of a serum field test which may assist the future diagnosis of TB.

Production of recombinant proteins in Escherichia coli using an N-terminal tag derived from sortase.

The use of Escherichia coli protein expression systems has many benefits, including the ease of propagation, amounts of protein that can be generated and cost. However, this host has some drawbacks due to difficulties in the production of soluble foreign proteins with their alternate codon usage bias, reductive cytoplasmic environment and lack of complex post-translational modifications. We have designed a novel fusion protein tag derived from the sequence of sortase (SrtA) which we have named Solubility 'eNhancing'Ubiquitous Tag (SNUT). Here we demonstrate its application and effectiveness as an N-terminal fusion tag for the expression and purification of proteins that could not be effectively produced with other tags. We show this tag can be utilized for the purification of proteins through both native and refolding immobilized metal ion chromatography and in combination with an anti-SNUT monoclonal antibody, can also be used as a detection tag. This tag may prove useful in circumventing expression and purification issues with the production of proteins in bacterial expression hosts.

Multiplex immunoassay for serological diagnosis of Mycobacterium bovis infection in cattle.

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.

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment.

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.

Inhibition of cathepsin L-like proteases by cathepsin V propeptide.

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.