Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells.
FcgammaRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting FcgammaRIII 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-FcgammaRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgammaRIIIB extra-cellular domains. These sdAbs bind FcgammaRIIIA+ NK cells and FcgammaRIIIB+ polymorphonuclear cells, but not FcgammaRI+ or FcgammaRII+ cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgammaRIII 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 FcgammaRIII with a K(D) in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgammaRIIIA 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-FcgammaRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgammaRIII killer cells to target and destroy tumor cells.
Behar
Ghislaine
G
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS, UPR9027, GDR2352, 31 chemin Joseph Aiguier, F-13402 Marseille Cedex 20, France.
Sibéril
Sophie
S
Groulet
Agnès
A
Chames
Patrick
P
Pugnière
Martine
M
Boix
Charlotte
C
Sautès-Fridman
Catherine
C
Teillaud
Jean-Luc
JL
Baty
Daniel
D
D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology* / Q000302:isolation & purification*; D000915:Antibody Affinity; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D002161:Camelids, New World / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005260:Female; D058851:GPI-Linked Proteins; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D007376:Interleukin-2 / Q000276:immunology / Q000378:metabolism; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D017452:Receptors, IgG / Q000276:immunology* / Q000378:metabolism
D018033:Antibodies, Bispecific; D015703:Antigens, CD; D000939:Epitopes; C496124:FCGR3A protein, human; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D007376:Interleukin-2; D017452:Receptors, IgG; D007371:Interferon-gamma