Effect of a single amino acid mutation on the activating and immunosuppressive properties of a "humanized" OKT3 monoclonal antibody.
The binding specificity of the murine OKT3 has been transferred into a human antibody framework to reduce its immunogenicity. This "humanized" anti-CD3 mAb (gOKT3-5) was previously shown to retain, in vitro, all the properties of native OKT3, including T cell activation, which has been correlated, in vivo, with the severe side effects observed in transplant recipients after the first administration of the mAb. T cell activation is thought to be triggered by the cross-linking mediated by the antibodies between T cells and Fc receptor-bearing cells. In this study, we introduced a single amino acid mutation from a leucine to a glutamic acid at position 235 in the Fc receptor binding segment of the gOKT3-5 mAb to produce Glu-235 mAb. This mutation generated a 100-fold decrease in the affinity of the antibody for the Fc receptor on U937 cells, without affecting Ag binding. In parallel, we observed a marked reduction in the T cell activation triggered by the mAb (proliferation, cell surface expression of early activation markers including Leu 23 and IL-2R, and release of TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF). In contrast, the mutated mAb retained suppressive properties similar to the gOKT3-5 mAb, as assessed by significant modulation of the T cell receptor complex and suppression of Ag-specific CTL activity. We conclude that this anti-CD3 mAb bearing a single amino acid mutation in its Fc portion retains important immunosuppressive properties, while exhibiting significantly less T cell activation than OKT3 in vitro. This drug might achieve potent immunosuppression while minimizing acute toxicity in vivo and thus be useful in transplantation as well as in autoimmune diseases.
Alegre
M L
ML
Ben May Institute, Committee on Immunology, University of Chicago, IL 60637.
Collins
A M
AM
Pulito
V L
VL
Brosius
R A
RA
Olson
W C
WC
Zivin
R A
RA
Knowles
R
R
Thistlethwaite
J R
JR
Jolliffe
L K
LK
Bluestone
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000943:Antigens, Differentiation / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D001666:Binding Sites, Antibody; D017252:CD3 Complex; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006801:Humans; D007165:Immunosuppression Therapy; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000096:biosynthesis; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D016853:Muromonab-CD3 / Q000737:chemistry* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D011961:Receptors, Fc / Q000378:metabolism; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000943:Antigens, Differentiation; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D016853:Muromonab-CD3; D011948:Receptors, Antigen, T-Cell; D011961:Receptors, Fc; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor