Tuberculosis (TB) is a major human disease of significant public health importance with an estimated 9.27 million new cases and 1.77 million deaths in 2007 alone [1]. Current methods for the diagnosis of tuberculosis are either costly, e.g. PCR or IFN release assays, or slow, e.g., sputum culture, or insensitive, e.g., X-rays, sputum smears. However, immunoassays such as ELISA and immunofluorescence (IF) offer a rapid, sensitive and specific means of diagnosis. However, such assays require a suitable antigen expressed by M. Tuberculosis for detection plus the generation of high affinity monoclonal antibodies of suitable specificity.
Lipoarabinomannan (LAM) is a membrane bound glycolipid that has been found to be present in the urine of TB patients [2, 3]. Structural studies on mycobacterial lipoarabinomannan have shown that LAM secreted by pathogenic mycobacteria such as M. avium, M. leprae, M. bovis and M. tuberculosis all have a mannose sugar capping motif (hence the term ManLAM) while other non-pathogenic mycobacteria have different capping motifs such as phosphoinositide for M. smegmatis (PILAM) and no caps for M. chelonae (AraLAM) [4]. LAM has a core structure consisting of a mannosyl-phosphatidyl-myo-inositol lipid anchor attached to a highly branched polysaccharide [4]. The core lipid anchor has up to four fatty acid chains which are thought to anchor the LAM in the plasma membrane of the bacterium. LAM can be extracted from crude cell wall fraction using ethanol solvent or boiling with SDS, indicating that it is tightly integrated but not covalently bound to the cell wall [24]. The branched polysaccharide consists of a repeating α1-6 mannose backbone with α1-5 arabinose branches which are decorated with secondary arabinose branches with α1-3,5 and β1-2 linkages. These secondary branches terminate with capping motifs that vary between different groups of mycobacteria. At least three different capping motifs have been described: a phosphoinositide cap in fast growing mycobacteria such as M. fortuitum and M. smegmatis, a mannose cap with up to three α1-2 linked mannose residues in slow-growing mycobacteria, which include all major pathogenic species such as M. tuberculosis, M. bovis, M. leprae and M. avium. In addition, the fast growing mycobacterial species M. chelonae has been found to have no sugar caps on its secondary arabinose branches [4].
Antibodies are useful diagnostic and therapeutic tools and various techniques such as animal immunization, monoclonal hybridomas and antibody phage display have been developed and applied to produce a wide range of antibodies with excellent specificities. Phage display is a more recent development of recombinant technology that allows for the generation of specific monoclonal antibodies without the need for an in vivo immune system response. It involves the display of a diverse antibody collection, either synthetically generated or obtained either from naturally-derived naïve or immune antibody repertoires, on filamentous phages which package the genetic sequence of their individual antibodies. Repeated selection on the desired target results in enrichment for specific antibody-phage clones from which the coding sequences can be recovered and use to produce recombinant antibodies. Another advantage of this system is that selection of specific antibodies with particular characteristics such as slow off-rates or fast on-rates or binding at extremes of pH or temperature, which is not possible in an in vivo immunization, can be carried out [38].
Thus, there is a need for antibodies with specificity for the mannose caps of ManLAM that have high diagnostic specificity as well as the ability to block the immunomodulatory activity of mannose caps.
USPTO Backgrounds
USPTO Backgrounds