Inhibition of IgE-dependent histamine release from human peripheral blood basophils by humanized Fab fragments that recognize the membrane proximal domain of the human Fc epsilon RI alpha-chain.
BACKGROUND
Inhibition of the interaction between IgE and the alpha-chain of Fc epsilon RI (Fc epsilon RI alpha) is a straightforward strategy to develop therapeutic reagents for IgE-mediated allergic diseases.


OBJECTIVE
The purpose of this study is the humanization of CRA2 and/or CRA4, mouse anti-human Fc epsilon RI alpha monoclonal antibodies (mAbs) which recognize the IgE-binding membrane proximal immunoglobulin-like domain of Fc epsilon RI alpha.


METHODS
The two mAbs were humanized by CDR grafting onto human V region frameworks encoded by human germline V and J genes. The activities of the recombinant antibodies to bind Fc epsilon RI alpha and inhibit IgE binding to Fc epsilon RI alpha were analyzed by flow cytometry and ELISA. Human peripheral blood basophils were pretreated with the Fab fragments of the humanized CRA2 and stimulated with IgE and an anti-IgE polyclonal antibody. The released histamine was measured.


RESULTS
The humanized CRA2 had almost the same activities of binding and inhibition of IgE binding to Fc epsilon RI alpha as the original mouse CRA2. Although the Fc epsilon RI-binding activity was maintained following humanization of the CRA4 light chain V region, it was lost by the humanization of the CRA4 heavy chain V region. Pretreatment of human peripheral blood basophils with the Fab fragments of the humanized CRA2 inhibited their subsequent degranulation activated by cross-linking of the Fc epsilon RI.


CONCLUSIONS
In the humanized CRA2, all amino acid residues except CDR are replaced with the residues encoded by human germline genes. The humanization of CRA2 might be an important step in the development of immunotherapy to manipulate the IgE network in which mast cells, basophils, and various types of Fc epsilon RI alpha expressing cells are involved.
Takai
T
T
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan.
Yuuki
T
T
Ra
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D001491:Basophils / Q000276:immunology*; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D017455:Receptors, IgE / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E