Conrad En Zuo Chan

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

Abstract Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development.

The role of phage display in therapeutic antibody discovery

Using phage-display to discover and isolate therapeutic antibodies

Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required.

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis.

Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 105 CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.

The diagnostic targeting of a carbohydrate virulence factor from M.Tuberculosis

The current clinical management of TB is complicated by the lack of suitable diagnostic tests that can be employed in infrastructure and resource poor regions. The mannose-capped form of lipoarabinomannan (ManLAM) is unique to the surface envelope of slow-growing, pathogenic mycobacteria such as M.tuberculosis (M.tb) and facilitates passive invasion of mononuclear phagocytes. The detection of this virulence factor in urine, sputum and serum has engendered interest in its employment as a biomarker for M.tb infection. In this study, we utilize a subtractive screening methodology to engineer the first high affinity recombinant antibody (My2F12) with exquisite specificity for the α1-2 mannose linkages enriched in ManLAM from M.tb. My2F12 binds to pathogenic mycobacterial species but not fast growing non-pathogenic species. Testing on matched urine and serum samples from TB patients indicates that My2F12 works in patient cohorts missed by other diagnostic methodologies.

Generation and Characterization of a Novel Recombinant Antibody Against 15-Ketocholestane Isolated by Phage-Display

The employment of monoclonal antibodies (Mabs) to identify disease-associated biomarkers in clinical samples represents the underlying principle for many diagnostic tests. To date, these have been principally developed for protein targets with few reported applications for lipids due to their hydrophobicity and poor immunogenicity. Oxysterols represent a family of lipids implicated in diverse human diseases where Mab-based detection assays could have a profound effect on their utility as clinical biomarkers. These are usually identified in patients’ samples by mass- spectrometry based approaches. Here, we describe an antibody phage-library based screening methodology for generating a recombinant monoclonal antibody (RAb) targeting the oxysterol-15-ketocholestane (15-KA), a lipid implicated in multiple sclerosis and Autoimmune Encephalomyelitis (EAE). The antibody is highly specific for 15-KA and shows little or no binding activity for other closely related oxysterols. We employ RAb2E9 to address the controversy over whether 15-KA is a true biomarker for MS/EAE and show that 15-KA is undetectable in serum taken from mice with EAE using antibody based detection methodologies; a finding confirmed by mass-spectrometry analysis. This study demonstrates the technical feasibility of using phage display to isolate highly specific antibodies against poorly immunogenic, small molecule lipids.

The Molecular Engineering of an Anti-Idiotypic Antibody for Pharmacokinetic Analysis of a Fully Human Anti-Infective.

Anti-idiotype monoclonal antibodies represent a class of reagents that are potentially optimal for analyzing the pharmacokinetics of fully human, anti-infective antibodies that have been developed as therapeutic candidates. This is particularly important where direct pathogen binding assays are complicated by requirements for biosafety level III or IV for pathogen handling. In this study, we describe the development of a recombinant, anti-idiotype monoclonal antibody termed E1 for the detection of a fully human, serotype-specific, therapeutic antibody candidate for the BSLIII pathogen Dengue virus termed 14c10 hG1. E1 was generated by naïve human Fab phage library panning technology and subsequently engineered as a monoclonal antibody. We show that E1 is highly specific for the fully-folded form of 14c10 hG1 and can be employed for the detection of this antibody in healthy human subjects’ serum by enzyme linked immunosorbent assay. In addition, we show that E1 is capable of blocking the binding of 14c10 hG1 to dengue virus serotype 1. Finally, we show that E1 can detect 14c10 hG1 in mouse serum after the administration of the therapeutic antibody in vivo. E1 represents an important new form of ancillary reagent that can be utilized in the clinical development of a therapeutic human antibody candidate.

Enhancing vaccine antibody responses by targeting Clec9A on dendritic cells

Targeting model antigens (Ags) to Clec9A on DC has been shown to induce, not only cytotoxic T cells, but also high levels of Ab. In fact, Ab responses against immunogenic Ag were effectively generated even in the absence of DC-activating adjuvants. Here we tested if targeting weakly immunogenic putative subunit vaccine Ags to Clec9A could enhance Ab responses to a level likely to be protective. The proposed “universal” influenza Ag, M2e and the enterovirus 71 Ag, SP70 were linked to anti-Clec9A Abs and injected into mice. Targeting these Ags to Clec9A greatly increased Ab titres. For optimal responses, a DC-activating adjuvant was required. For optimal responses, a boost injection was also needed, but the high Ab titres against the targeting construct blocked Clec9A-targeted boosting. Heterologous prime-boost strategies avoiding cross-reactivity between the priming and boosting targeting constructs overcame this limitation. In addition, targeting small amounts of Ag to Clec9A served as an efficient priming for a conventional boost with higher levels of untargeted Ag. Using this Clec9A-targeted priming, conventional boosting strategy, M2e immunisation protected mice from infection with lethal doses of influenza H1N1 virus.Vaccine technology: targeting the vaccine to the immune systemDuration and intensity of vaccine response can be boosted using antibodies to target pathogen fragments to specific immune system cells. Dendritic cells exist to take fragments of infectious diseases and present them to the immune system, sparking host defenses. Now, researchers led by Monash University’s Mireille Lahoud, and Ken Shortman of the Walter and Eliza Hall Institute, have successfully used antibodies to target fragments of influenza and hand, foot and mouth disease directly to dendritic cell molecules, specifically chosen to elicit a prolonged immune response. Mice inoculated with the targeted vaccine were protected from lethal influenza exposure, whereas the hand, foot and mouth disease vaccine elicited promising, but less marked results. With further development, this technology could provide a vital boost to vaccines that offer poor immunity on their own.

TCR–like antibodies mediate complement and antibody-dependent cellular cytotoxicity against Epstein-Barr virus–transformed B lymphoblastoid cells expressing different HLA-A*02 microvariants

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV–transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.