Characterization of nanobodies binding human fibrinogen selected by E. coli display

Salema, Valencio; López-Guajardo, Ana; Gutiérrez, Carlos; Mencı́a, Mario; Fernández, Luis A.

doi:10.1016/j.jbiotec.2016.07.025

Cited by 18 publications

(31 citation statements)

References 33 publications

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“…In our experience, the number of cycles required for the isolation of high‐affinity Nb clones varies depending on the library, but usually two rounds of MACS biopanning are sufficient to observe a significant enrichment of antigen‐specific binders from immune libraries allowing isolation of high‐affinity Nb clones with K D s in the low nanomolar and subnanomolar range (i.e. 0.2–20 nM; Salema et al ., , ,b). Upon MACS biopannings, isolation of E. coli clones binding the antigen is performed by flow cytometry screening of individual colonies (e.g.…”

Section: E Coli Displaymentioning

confidence: 99%

“…streptavidin–PE) for the determination of antigen binding and specificity (Fig. ; Top panel; Salema et al ., , ,b). Nb display and antigen binding can be analysed simultaneously (double staining) or independently (single staining).…”

Section: E Coli Displaymentioning

confidence: 99%

“…We have tested the intimin E. coli display system with three different VHH immune libraries, two from dromedaries and one from llamas. Each library was obtained after immunization with antigens of different origin: a purified recombinant bacterial polypeptide (TirM; Salema et al ., ), purified human fibrinogen (Fib; Salema et al ., 2016a) and cells from the human tumour cell line A431 overexpressing the epidermal growth factor receptor (EGFR; Salema et al ., b). In all cases, the VHH gene segments were amplified from lymphocytes isolated from peripheral blood samples and cloned in frame with the Neae fragment in pNeae2 vector to obtain ~10 7 independent clones (Fig.…”

Section: E Coli Displaymentioning

confidence: 99%

“…Given the potential of Nbs for tumour therapy and in vivo tumour imaging, we evaluated E. coli display for the direct selection on live cells of Nbs against tumour‐associated cell surface antigens (Scott et al ., ; Tumeh et al ., ; Postow et al ., ; Redman et al ., ). In particular, we performed selection on live cells of Nbs binding human EGFR as a model tumour cell surface antigen (Salema et al ., b). Although MACS is a convenient method to select high‐affinity binders, it relies on the availability of purified antigen, which is not always practical or feasible.…”

Section: E Coli Displaymentioning

confidence: 99%

“…To screen for binders, we examined by bright‐field light microscopy the adhesion of 96 clones, picked from the second round of CellS, to Her14 (EGFR+) and NIH‐3T3 2.2 (EGFR‐) cells. Clones binding specifically Her14 cells were selected, identifying high‐affinity Nbs that bind specifically EGFR‐Fc by flow cytometry (Salema et al ., b). As high‐affinity binders were predominantly selected during the screening, this indicates that the high levels of display of Nbs on E. coli surface and of the target EGFR antigen on the plasma cell membrane of the transfected cell line had no significant avidity effect, which could have resulted in the frequent isolation of low‐affinity binders.…”

Section: E Coli Displaymentioning

confidence: 99%

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Escherichia coli surface display for the selection of nanobodies

Salema

Fernández

2017

Microbial Biotechnology

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SummaryNanobodies (Nbs) are the smallest functional antibody fragments known in nature and have multiple applications in biomedicine or environmental monitoring. Nbs are derived from the variable segment of camelid heavy chain‐only antibodies, known as VHH. For selection, libraries of VHH gene segments from naïve, immunized animals or of synthetic origin have been traditionally cloned in E. coli phage display or yeast display systems, and clones binding the target antigen recovered, usually from plastic surfaces with the immobilized antigen (phage display) or using fluorescence‐activated cell sorting (FACS; yeast display). This review briefly describes these conventional approaches and focuses on the distinct properties of an E. coli display system developed in our laboratory, which combines the benefits of both phage display and yeast display systems. We demonstrate that E. coli display using an N‐terminal domain of intimin is an effective platform for the surface display of VHH libraries enabling selection of high‐affinity Nbs by magnetic cell sorting and direct selection on live mammalian cells displaying the target antigen on their surface. Flow cytometry analysis of E. coli bacteria displaying the Nbs on their surface allows monitoring of the selection process, facilitates screening, characterization of antigen‐binding clones, specificity, ligand competition and estimation of the equilibrium dissociation constant (KD).

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Section: E Coli Displaymentioning

confidence: 99%

Section: E Coli Displaymentioning

confidence: 99%

Section: E Coli Displaymentioning

confidence: 99%

Section: E Coli Displaymentioning

confidence: 99%

Section: E Coli Displaymentioning

confidence: 99%

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Escherichia coli surface display for the selection of nanobodies

Salema

Fernández

2017

Microbial Biotechnology

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Camelid‐derived single‐chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications

Peyron

Kizlik-Masson

Dubois

et al. 2020

Research and Practice in Thrombosis and Haemostasis

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Hemostasis is a complex process involving the concerted action of molecular and vascular components. Its basic understanding as well as diagnostic and therapeutic aspects have greatly benefited from the use of monoclonal antibodies. Interestingly, camelid‐derived single‐domain antibodies (sdAbs), also known as V H H or nanobodies, have become available during the previous 2 decades as alternative tools in this regard. Compared to classic antibodies, sdAbs are easier to produce and their small size facilitates their engineering and functionalization. It is not surprising, therefore, that sdAbs are increasingly used in hemostasis‐related research. In addition, they have the capacity to recognize unique epitopes unavailable to full monoclonal antibodies. This property can be used to develop novel diagnostic tests identifying conformational variants of hemostatic proteins. Examples include sdAbs that bind active but not globular von Willebrand factor or free factor VIIa but not tissue factor–bound factor VIIa. Finally, sdAbs have a high therapeutic potential, exemplified by caplacizumab, a homodimeric sdAb targeting von Willebrand factor that is approved for the treatment of thrombotic thrombocytopenic purpura. In this review, the various applications of sdAbs in thrombosis and hemostasis‐related research, diagnostics, and therapeutic strategies will be discussed.

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