title: Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes.
abstract: The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Connelly
authors.forename: M S
authors.initials: MS
authors.lastname: Hayden
authors.forename: J K
authors.initials: JK
authors.lastname: Scholler
authors.forename: T T
authors.initials: TT
authors.lastname: Tsu
authors.forename: B
authors.initials: B
authors.lastname: Dupont
authors.forename: J A
authors.initials: JA
authors.lastname: Ledbetter
authors.forename: S B
authors.initials: SB
authors.lastname: Kanner
mesh_terms: D000069594:Abatacept; D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000494:pharmacology; D000918:Antibody Specificity / Q000235:genetics; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive / Q000276:immunology; D018801:CD2 Antigens / Q000276:immunology*; D019556:COS Cells; D060908:CTLA-4 Antigen; D002118:Calcium / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006801:Humans; D018796:Immunoconjugates; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D018801:CD2 Antigens; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018984:Epitopes, T-Lymphocyte; D018796:Immunoconjugates; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008934:Mitogens; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D000069594:Abatacept; D002118:Calcium