tNOX proteins are absent or reduced to below the limits of detection from sera of healthy volunteers or patients with diseases other than cancer. Circulating tNOX has been detected in sera of more than 500 cancer patients representing all major forms of human cancer including leukemias and lymphomas. All tNOX isoforms share a common antigenic determinant recognized by a tNOX-specific monoclonal antibody and a corresponding single chain variable region (scFv) fragment expressed in bacteria (Cho et al., 2002). The latter was produced from tNOX-specific IgG rescued from the monoclonal antibody-producing hybridoma cells. Additionally, a phage display antibody carrying a myc tag was produced using bacterially expressed 17 kDa N- terminus of the expressed tNOX protein as template but with utility limited to Western blots. Both the phage display antibody and the scFv recombinant antibody exhibit nearly absolute specificity for tNOX isoforms and a high degree of sensitivity on Western blots but for reasons of epitope availability do not distinguish cancer from non-cancer in an ELISA format. Thus the need to develop new antibodies targeted to ELISA-accessible protein domains amenable to ELISA development is currently underway at NOX Technologies.
The presence of the tNOX protein has been demonstrated in a number of human tumor tissues and xenografts. However, serum analyses suggest a much broader association with cancer. tNOX proteins are ectoproteins reversibly bound at the outer leaflet of the plasma membrane (Morré, 1995). As is characteristic of other examples of ectoproteins (sialyl and galactosyl transferases, dipeptidylamino peptidase IV, etc.), the tNOX proteins are shed (Morré et al., 1996; Wilkinson et al., 1996). They appear in soluble form in conditioned media of cultured cells and in patient sera. The tNOX-isoforms from sera of cancer patients exhibits the same degree of drug responsiveness as do the membrane-associated forms (Morré and Reust, 1997; Morré et al., 1997). In contrast, no drug-responsive NOX activities were found with sera from healthy volunteers or sera from patients with diseases other than cancer. As such, the antitumor-responsive tNOX activity represents the first reported cell surface change absent from non-cancer cells and potentially associated with most, if not all, forms of human cancer.
tNOX specificity is indicated from lack of a drug-responsive NOX activity in normal tissues, lack of a serum form of tNOX in healthy volunteers and patients with disorders other than cancer and indications from tNOX-specific antisera. Findings to date with both cultured cells and sera offer no evidence for a tNOX cross-reactive protein other than with cancer. tNOX has been cloned (Chueh et al., 2002) using a tNOX isoform-specific monoclonal antibody to a common tNOX epitope that includes the cancer drug-binding site (Cho et al., 2002) from which our pan tNOX isoform-specific scFv recombinant antibody is derived.
The circulating tNOX markers are isoforms constituting the tNOX sub family of ECTO-NOX proteins that contribute to the unregulated growth of cancer cells. They appear to be structurally related byt distinct splice variants (Tang et al. 2007) none of which appear to be produced by non-cancer cells. Yet each major form of human cancer appear to be characterized by a unique isoform or pattern of isoforms permitting not only early detetion but diagnosis as to tissue of origin as well.
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