Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes

• Published in: International immunology Vol. 10; no. 12; pp. 1863 - 1872
• Main Authors: Connelly, R J, Hayden, M S, Scholler, J K, Tsu, T T, Dupont, B, Ledbetter, J A, Kanner, S B
• Format: Journal Article
• Language: English
• Published: England 01.12.1998
• Subjects: Abatacept; Amino Acid Sequence; Animals; Antibodies, Bispecific - biosynthesis; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - genetics; Antibodies, Bispecific - pharmacology; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - pharmacology; Antibody Specificity - genetics; Antigens, CD; Antigens, Differentiation - pharmacology; Base Sequence; Binding, Competitive - immunology; Calcium - metabolism; CD2 Antigens - immunology; COS Cells; CTLA-4 Antigen; Epitopes, T-Lymphocyte - immunology; Humans; Immunoconjugates; Immunoglobulin Fragments - biosynthesis; Immunoglobulin Fragments - chemistry; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - pharmacology; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Heavy Chains - metabolism; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - metabolism; Intracellular Fluid - metabolism; Lymphocyte Activation - immunology; Lymphocyte Culture Test, Mixed; Mitogens - pharmacology; Molecular Sequence Data; Recombinant Fusion Proteins - chemical synthesis; Recombinant Fusion Proteins - immunology; T-Lymphocytes - immunology
• ISSN: 0953-8178; 1460-2377
• DOI: 10.1093/intimm/10.12.1863

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.