| 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. |
| Keywords: |
controlled study; human cell; protein domain; lymphocyte proliferation; t lymphocyte; t-lymphocytes; animals; signaling; mitogenesis; calcium; cos cells; monoclonal antibody; molecular cloning; cd2 antigen; lymphocyte activation; lymphocyte culture test, mixed; antibodies, monoclonal; immunoglobulin variable region; amino acid sequence; immunoglobulin heavy chains; molecular sequence data; recombinant fusion proteins; base sequence; binding site; antibody specificity; antibodies; antigens, cd; immunoglobulin g1; immunoglobulin g3; cytotoxic t lymphocyte antigen 4; epitopes, t-lymphocyte; t lymphocyte activation; mixed lymphocyte reaction; antigens, differentiation; t lymphocytes; mitogens; binding, competitive; immunoglobulin fragments; immunoconjugates; calcium ion; calcium mobilization; antibodies, bispecific; antigens, cd2; humans; human; priority journal; article; accessory cell; cellular activation; intracellular fluid; co-stimulatory molecules
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