Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes [In Process Citation]

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinctepitopes, induces proliferation of resting human T cells. The mitogenicactivity of this mAb mixture depends upon accessory cells and the 9-1 mAbFc domain. To further study the functional properties of these mAb, theirvariable regions were cloned and expressed as monospecific single- chain Fv(scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novelbispecific scFvIg was constructed by cloning the two monospecific scFvbinding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of thecorresponding parental mAb, while the bispecific scFvIg exhibited bindingactivity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIgand 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg werenon-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc.Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenicand was a more potent mitogen than the mAb mixture, but was accessory celldependent. Unlike the combination of mAb, the bispecific reagent did notdirectly mobilize calcium in T cells. In comparison to the mAb mixture,bispecific 9.6/9- 1 scFvIg-mediated stimulation of a mixed lymphocytereaction was significantly more resistant to inhibition of the CD28co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show thatexpression of the 9.6 and 9-1 binding sites together on a bispecific scFvIgincreased the mitogenic properties of the mAb and altered the degree ofaccessory cell signals required for T cell activation.