Expression of humanized Fab fragments that recognize the IgE-binding domain of human Fc(epsilon)RIalpha in COS and CHO cells.
Interfering with the binding of IgE to high-affinity IgE receptor alpha chain (Fc(epsilon)RIalpha) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human Fc(epsilon)RIalpha inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human Fc(epsilon)RIalpha Fabs without papain-digestion of the whole antibody. Four Fabs with different C-termini of CH1 were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-Fc(epsilon)RIalpha binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human Fc(epsilon)RIalpha will provide useful information for the design of therapeutic reagents for allergy and asthma.
Takai
T
T
Bioscience Research & Development Laboratory, Asahi Breweries, Ltd., Midori Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan. toshiro.takai@asahibeer.co.jp
Okumura
K
K
Ra
C
C
Yokota
T
T
Okumura
Y
Y
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary; D017455:Receptors, IgE / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D016895:Culture Media, Serum-Free; D007140:Immunoglobulin Fab Fragments; D017455:Receptors, IgE; D007073:Immunoglobulin E