title: OKT3 F(ab')2 fragments--retention of the immunosuppressive properties of whole antibody with marked reduction in T cell activation and lymphokine release.
abstract: Recent studies in mouse and man indicate that the first dose response to anti-CD3 mAbs likely results from in vivo T cell activation and concomitant lymphokine release. One approach toward amelioration of these effects involves the use of nonactivating digest fragment preparations of anti-CD3 mAbs. In the present study whole and F(ab')2 fragments of OKT3 were prepared and assayed for their immune-activating and -suppressing effects on human peripheral blood mononuclear cells. Immunosuppressive effects were evaluated by quantitation of TCR modulation and coating, and by inhibition of CTL activity. Whole mAb and F(ab')2 fragments both effectively coated the TCR complex. However, whole mAb was more efficient at modulating the TCR complex, suggesting that modulation is enhanced by FcR interactions. Whole and F(ab')2 fragments of OKT3 were equally efficacious in suppressing CTL activity. Immune activation was evaluated by quantitation of proliferation, activation marker expression (IL-2R and Leu-23), and lymphokine release (TNF-alpha, gamma-IFN, and GM-CSF). Rigorously purified F(ab')2 preparations demonstrated minimal T cell activation, suggesting TCR and macrophage FcR crosslinking as necessary. Whole OKT3 mAb induced expression of IL-2R and Leu-23 activation markers on the majority of CD4+ and CD8+ cells at mAb concentrations as low as 1 ng/ml, whereas F(ab')2 fragments induced detectable, but markedly reduced expression of these markers only at mAb concentrations greater than or equal to 100 ng/ml. Similarly, whole mAb induced release of TNF-alpha, gamma-IFN, and GM-CSF at low mAb concentrations, whereas F(ab')2 fragments induced detectable (though markedly reduced) levels of TNF-alpha only. However, increasing degrees of contamination with whole antibody resulted in increasing mitogenic potency of the F(ab')2 preparation, which in some cases, was actually enhanced compared with that observed with whole mAb alone. In conclusion, these studies indicate that OKT3 F(ab')2 digest fragments are markedly less potent than whole mAb in inducing T cell activation, yet they retain significant immunosuppressive effects. However, meticulous purification of F(ab')2 digest fragment preparations will likely be required to avoid T cell and macrophage activation following in vivo administration.
authors.affiliation: Department of Surgery, Ben May Institute, University of Chicago School of Medicine, Illinois 60637.
authors.forename: E S
authors.initials: ES
authors.lastname: Woodle
authors.forename: J R
authors.initials: JR
authors.lastname: Thistlethwaite
authors.forename: I A
authors.initials: IA
authors.lastname: Ghobrial
authors.forename: L K
authors.initials: LK
authors.lastname: Jolliffe
authors.forename: F P
authors.initials: FP
authors.lastname: Stuart
authors.forename: J A
authors.initials: JA
authors.lastname: Bluestone
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology; D015704:CD4 Antigens / Q000502:physiology; D016827:CD8 Antigens; D004305:Dose-Response Relationship, Drug; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000378:metabolism; D007371:Interferon-gamma / Q000096:biosynthesis / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology*; D008222:Lymphokines / Q000378:metabolism*; D010434:Pepsin A / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D013601:T-Lymphocytes / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D015704:CD4 Antigens; D016827:CD8 Antigens; D007140:Immunoglobulin Fab Fragments; D008222:Lymphokines; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D010434:Pepsin A