A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies

Jones, Martina L.; Seldon, Therese; Smede, Matthew; Linville, Ashleigh; Chin, David Y.; Barnard, Ross; Mahler, Stephen M.; Munster, David J.; Hart, Derek N. J.; Gray, Peter P.; Munro, Trent P.

doi:10.1016/j.jim.2010.02.001

Cited by 48 publications

(57 citation statements)

References 10 publications

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“…To reformat the scFv fragments to whole, fully human IgG, the variable regions for both the heavy and kappa light chains of the rPfHRP2 specific scFv clones D2 and F9 were PCR amplified from the phagemid vectors and cloned into SacI-linearized ReformAb vectors (Acyte Biotech, Australia) as previously described [26]. …”

Section: Methodsmentioning

confidence: 99%

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Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2

Leow

Jones

Cheng³

et al. 2014

Malar J

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BackgroundEarly and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs.MethodsIn this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized.ResultsTwo unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10−11 M, followed by control mAb C1-13 at 1.03 × 10−10 M and mAb D2 at 3.05 × 10−10 M.ConclusionsOverall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.

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Section: Methodsmentioning

confidence: 99%

“…The expression and purification of mAbs D2 and F9 was undertaken as previously described [26]. Briefly, the heavy and light chain plasmids were co-transfected into suspension-adapted Chinese hamster ovary (CHO) cells after mixing of plasmids with polyethylenimine (PEI).…”

Section: Methodsmentioning

confidence: 99%

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2

Leow

Jones

Cheng³

et al. 2014

Malar J

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“…Not all LIC methods are discussed in detail in this review, for example, the In-Fusion kit 6,31 which is a patent kit sold by Takara Bio Inc. (Dalian, China), and the nicking DNA endonuclease, in which the long sticky ends are created using a restriction enzyme Nt.BbvCI 32 . The readers can refer to the cited references for more information.…”

Section: Discussionmentioning

confidence: 99%

Ligation independent cloning: an efficient tool for high-throughput cloning and modular cDNA assembly

Liu¹

2013

OA Biotechnology

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IntroductionTo overcome the shortcomings of traditional cloning methods, ligasefree methods or ligation independent methods were developed to provide more choices for technicians. In this article, the history, mechanism and application of four in vitro ligasefree methods are reviewed in detail: (i) ligation independent cloning, (ii) gateway recombinational cloning, (iii) uracil-DNA glycosylase and (iv) restriction free cloning. Conclusion These ligation independent cloning tools are simple, fast and efficient tools for high-throughput cloning or long expression vector assembly.

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“…The variable regions of motavizumab and D25 antibodies were cloned and expressed in recombinant form. Briefly, the variable domains of the antibodies were cloned into separate plasmids encoding the heavy-and light-chain human IgG backbones described by Jones et al (37). Vectors were transfected into CHO cells in suspension culture, and supernatant was harvested 7 days later for secreted antibody, which was purified by protein A chromatography and buffer exchanged into PBS as previously described (38).…”

Section: Methodsmentioning

confidence: 99%

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

Bermingham

Chappell

Watterson

et al. 2018

J Virol

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Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F 1 and F 2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic ␣-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F 2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F 2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.KEYWORDS respiratory syncytial virus, fusion, membrane fusion, mutagenesis, heptad repeat R espiratory syncytial virus (RSV) is widely considered the most significant viral pediatric pathogen worldwide. Belonging to the Pneumovirinae family, RSV causes substantial morbidity and mortality worldwide, with an estimated 33.8 million acute lower respiratory tract infections per year in children under 5 years of age, and has been linked to almost 200,000 deaths annually, 99% of which are in developing countries (1, 2). RSV is also recognized as an important pathogen of high-risk adult and elderly populations (3). Despite this, no targeted drug or vaccin...

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A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies

Cited by 48 publications

References 10 publications

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2

Ligation independent cloning: an efficient tool for high-throughput cloning and modular cDNA assembly

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

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