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.

Novel tetravalent and bispecific IgG‐like antibody molecules combining single‐chain diabodies with the immunoglobulin γ1 Fc or CH3 region

“…Furthermore, since the two binding sites are present in one single‐chain diabody molecule, bispecific IgG‐like molecules are formed by simple Fc‐mediated homodimerisation giving rise to a homogeneous population of molecules. A similar approach to generate bispecific IgG‐like molecules has recently been described fusing two scFv fragments arranged in tandem to the IgG1 Fc region [30]. These bispecific antibody molecules recognising two epitopes on CD2 exhibited increased mitogenic properties compared to the parental monoclonal antibodies.…”

Alefacept selectively promotes NK cell‐mediated deletion of CD45R0+ human T cells

“…Binding of LFA-3 to CD2 is independent of TCR stimulation, but the interaction facilitates the T cell recognition process and optimizes subsequent antigen-specific T cell activation [3]. CD2-mediated signaling requires TCR expression unless the CD2 expression level is very high [4,5]. CD2 is important in augmenting CD3-mediated proliferation and signaling [6], so providing costimulatory function for resting T cells during initial activation [7,8].…”

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

“…13 Other bispecific antibody formats, termed BiTE (bispecific T-cell engager) and DART (dual-affinity re-targeting), are based on the covalent linkage of two single-chain antibodies by a peptide linker 14 and a disulfide bridge, 15 respectively. For in vivo use, various formats of scFv fusion proteins, such as scFv-IgG, 16 scFv-Fc-scFv 17 and tandem scFv-Fc, 18 were developed because the Fc domain provided both prolonged pharmacokinetic half-life and Fc-mediated effector function.…”