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PEDS Advance Access originally published online on December 11, 2007
Protein Engineering Design and Selection 2008 21(1):1-10; doi:10.1093/protein/gzm064
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Isolation and characterization of anti-Fc{gamma}RIII (CD16) llama single-domain antibodies that activate natural killer cells

Ghislaine Behar1, Sophie Sibéril2,3,4, Agnès Groulet1, Patrick Chames1, Martine Pugnière5, Charlotte Boix2,3,4, Catherine Sautès-Fridman2,3,4, Jean-Luc Teillaud2,3,4,6,7 and Daniel Baty1,6,7

1 Laboratoire d’Ingénierie des Systèmes Macromoléculaires, CNRS, UPR9027, GDR2352, 31 chemin Joseph Aiguier, F-13402 Marseille Cedex 20, France 2 INSERM, U872, CNRS GDR2352, 15 rue de l’école de médecine, F-75006 Paris, France 3Centre de Recherche des Cordeliers, Université Pierre et Marie Curie – Paris 6 UMRS872, Paris F-75006, France 4 Université Paris Descartes, UMRS872, Paris, F-75006 France 5 Centre de Pharmacologie et Innovation dans le Diabète, Faculté de Pharmacie, CNRS, UMR5232, 15 Avenue Charles Flahault, BP 14491, F-34093 Montpellier Cedex 5, GDR2352, France

7 To whom correspondance should be addressed. E-mail: baty{at}ibsm.cnrs-mrs.fr (D.B.)/ jean-luc.teillaud{at}crc.jussieu.fr (J.-L.T.)

Fc{gamma}RIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting Fc{gamma}RIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-Fc{gamma}RIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with Fc{gamma}RIIIB extra-cellular domains. These sdAbs bind Fc{gamma}RIIIA+ NK cells and Fc{gamma}RIIIB+ polymorphonuclear cells, but not Fc{gamma}RI+ or Fc{gamma}RII+ cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different Fc{gamma}RIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind Fc{gamma}RIII with a KD in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of Fc{gamma}RIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-{gamma} production, respectively. These anti-Fc{gamma}RIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit Fc{gamma}RIII killer cells to target and destroy tumor cells.

Keywords: CD16/Fc{gamma}RIII/llama/phage display/single-domain antibodies

Received June 26, 2007; revised September 4, 2007; accepted October 8, 2007.

6 J.-L.T. and D.B. are senior co-authors.


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