Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization.
Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.
Veri
Maria-Concetta
MC
MacroGenics Inc., Rockville, MD 20850, USA.
Gorlatov
Sergey
S
Li
Hua
H
Burke
Steve
S
Johnson
Syd
S
Stavenhagen
Jeffrey
J
Stein
Kathryn E
KE
Bonvini
Ezio
E
Koenig
Scott
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000378:metabolism; D015703:Antigens, CD / Q000097:blood* / Q000235:genetics / Q000276:immunology; D005838:Genotype; D006801:Humans; D007962:Leukocytes / Q000276:immunology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D017452:Receptors, IgG / Q000097:blood* / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D015398:Signal Transduction / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D015703:Antigens, CD; C080568:Fc gamma receptor IIA; C080571:Fc gamma receptor IIB; D017452:Receptors, IgG