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Santa Barbara, CA, United States

Beasley M.D.,Affinity Bio | Niven K.P.,Affinity Bio | Winnall W.R.,University of Melbourne | Kiefel B.R.,Affinity Bio
Biotechnology Journal

Conventional antibody surface display requires fusion protein export through at least one cellular membrane, constraining the yield and occasioning difficulties in achieving scaled production. To circumvent this limitation, we developed a novel cytoplasmic display platform, Retained Display (ReD), and used it to screen for human scFv frameworks that are highly soluble and stable in the bacterial cytoplasm. ReD, based on the retention of high-molecular weight complexes within detergent-permeabilized Escherichia coli, enabled presentation of exogenous targets to antibodies that were expressed and folded in the cytoplasm. All human λ and κ light chain family genes were expressed as IGHV3-23 fusions. Members of the λ subfamilies 1, 3 and 6 were soluble cytoplasmic partners of IGHV3-23. Contrary to previous in vivo screens for soluble reduced scFvs, the pairings identified by ReD were identical to the human germline sequences for the framework, CDR1 and CDR2 regions. Using the most soluble scFv scaffold identified, we demonstrated tolerance to CDR3 diversification and isolated a binding scFv to an exogenous protein target. This screening system has the potential to rapidly produce antibodies to target threats such as emerging infectious diseases and bioterror agents. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Winnall W.R.,University of Melbourne | Beasley M.D.,Affinity Bio | Center R.J.,University of Melbourne | Center R.J.,Burnet Institute | And 3 more authors.
Immunology and Cell Biology

Antibodies are one of our most useful biological tools. Indeed, improvements in antibody-based technologies have ushered in a new era of antibody-based therapeutics, research and diagnostic tools. Although improved technologies have led to the development of therapeutic antibodies for treatment of malignancies and inflammatory conditions, the use of advanced antibody technology in the therapy of viral infections is in its infancy. Non-human primate studies have demonstrated that antibodies against the HIV envelope can both prevent viral infection and control viremia. Despite the obvious potential of antibody therapies against HIV, there remain limitations in production and purification capacity that require further research. Recent advances in recombinant antibody technology have led to the development of a range of novel antibody fragments, such as single-domain nanobodies and bispecific antibodies, that are capable of targeting cancer cells to cytotoxic T cells. Novel antibody production techniques have also been designed, allowing antibodies to be obtained from non-mammalian cells, bovine colostrum and the periplasm and cytoplasm of bacteria. These advances may allow large-scale production of HIV antibodies that are capable of protecting against HIV infection or serving as therapeutics that reduce the need for life-long antiretroviral treatment. This review summarises recent advances in antibody-based technologies and discusses the possibilities and challenges of using these advances to design prophylactics and therapeutics against HIV. © 2014 Australasian Society for Immunology Inc. Source

Brummelhuis W.J.,University Utrecht | Joles J.A.,University Utrecht | Stam J.C.,University Utrecht | Adams H.,Affinity Bio | And 7 more authors.

Staphylococcus aureus produces the superantigen toxic shock syndrome toxin 1 (TSST-1). When the bacterium invades the human circulation, this toxin can induce life-threatening gram-positive sepsis. Current sepsis treatment does not remove bacterial toxins. Variable domains of llama heavy-chain antibodies (VHH) against toxic shock syndrome toxin 1 (α-TSST-1 VHH) were previously found to be effective in vitro. We hypothesized that removing TSST-1 with α-TSST-1 VHH hemofiltration filters would ameliorate experimental sepsis in pigs. After assessing in vitro whether timely removing TSST-1 interrupted TSST-1-induced mononuclear cell TNF-α production, VHH-coated filters were applied in a porcine sepsis model. Clinical course, survival, plasma interferon γ, and TSST-1 levels were similar with and without VHH-coated filters as were TSST-1 concentrations before and after the VHH filter. Plasma TSST-1 levels were much lower than anticipated from the distribution of the amount of infused TSST-1, suggesting compartmentalization to space or adhesion to surface not accessible to hemofiltration or pheresis techniques. Removing TSST-1 from plasma was feasible in vitro. However, the α-TSST-1 VHH adsorption filter-based technique was ineffective in vivo, indicating that improvement of VHH-based hemofiltration is required. Sequestration likely prevented the adequate removal of TSST-1. The latter warrants further investigation of TSST-1 distribution and clearance in vivo. © 2010 by the Shock Society. Source

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