Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies

Robert, Rémy; Noubhani, Abdelmajid; Jacobin, Marie-Josée; Santarelli, Xavier; Clofent‐Sanchez, Gisèle

doi:10.1016/j.jchromb.2004.10.038

Cited by 6 publications

(4 citation statements)

References 27 publications

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“…TEG4 scFv had been previously expressed in E coli . [ 27 ] Unfortunately, despite optimization tests leading to high yields of cytoplasmic production, proteins also frequently accumulated into inclusion bodies (data not shown). In bacterial systems, many scFv can be produced into the periplasmic space but they are obtained with a very low yield.…”

Section: Discussionmentioning

confidence: 99%

“…Aliquots of 10 8 PFA-fixed-washed-platelets/mL (10 μL) [ 27 ] non-activated (NA-PL) or activated with thrombin (A-PL) were incubated with 25 μg/mL of TEG4-2c scFv human antibody or PAC-1 commercial IgM antibody (BD Biosciences) [ 31 ] targeting the activated αIIbβ3 integrin overnight at 4°C. After two washes in PBS, a 30 min incubation with secondary Alexa Fluor 488 anti-6His or goat anti-mouse IgM antibodies (1:20) was performed for detection of respectively, the TEG4-2c scFv and the murine PAC-1 antibody.…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, despite improvements in fedbatch fermentation production, a proportion of inactive scFv remained in the total purified population. [ 27 ] From this point of view, the Pichia pastoris expression system is an attractive way due to its ability to secrete a large amount of recombinant protein in the supernatant, thereby facilitating the purification steps.…”

Section: Introductionmentioning

confidence: 99%

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A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

Vallet-Courbin¹,

Larivière²,

Hocquellet³

et al. 2017

PLoS ONE

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Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

Vallet-Courbin¹,

Larivière²,

Hocquellet³

et al. 2017

PLoS ONE

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show abstract

“…We (58,59) and others (60)(61)(62) have taken advantage of the existence of different conformational states of GPIIb/IIIa to develop an alternative, unique strategy that targets only activated platelets. As scFv phage display permits an in-vitro imitation of the natural immunisation process, we used a subtractive panning strategy to select antibodies against discretely differing epitopes (8).…”

Section: Scfvs Against the Platelet Integrin Gpiib/iiiamentioning

confidence: 99%

Single-chain antibodies as diagnostic tools and therapeutic agents

et al. 2009

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SummaryOver three decades after the generation of the first mouse monoclonal antibodies by Kohler and Milstein, recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. Technology improvements have provided several approaches to manufacturing human antibodies with high affinity for biologically relevant targets. Approximately 300 development programs for therapeutic antibodies have been reported in industrial and academic laboratories, and this clearly demonstrates the expectations towards antibody technology. Keywords Antibody fragments, imaging, thrombosis, inflammation, phagedisplayAntibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as one of the smallest possible format for recombinant antibodies and their use as diagnostic tools and therapeutic agents. We describe the structure, selection and production of single-chain antibodies. Furthermore, we review current applications of antibody fragments focusing on thrombus targeting using fibrin-and plateletspecific single-chain antibodies as well as describing novel noninvasive imaging approaches for the diagnosis of thrombosis and inflammation.

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Purification of single-chain antibody fragments exploiting pH-gradients in simulated moving bed chromatography

Cristancho

Seidel‐Morgenstern

2016

Journal of Chromatography A

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Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies

Cited by 6 publications

References 27 publications

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

Single-chain antibodies as diagnostic tools and therapeutic agents

Purification of single-chain antibody fragments exploiting pH-gradients in simulated moving bed chromatography

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