Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2-and CD16-expressing cells at an optimum concentration of 100 nM. We introduce novel measurements with supported planer bilayers, from which key twodimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2-CD58 interaction. Alefacept mediated maximal adhesion of CD2 ؉ T cells to CD16B, an Fc receptor, in planar bilayers at 500 nM. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/m 2 and two-dimensional association constants of 3.1 and 630 m 2 for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation.Immunoadhesins are biopharmaceuticals that take the basic framework of antibodies and replace the antigen-binding domain with the ectodomain of adhesion molecules (1, 2). The common fragment of IgG (Fc) portion is thought to link to immune effector mechanisms to destroy cancer cells and or over-reactive immune cells. One example of this approach is the drug alefacept, which combines the CD2 binding domain of the adhesion molecule CD58 (LFA-3) with human IgG1 Fc in a single polypeptide chain. Alefacept is approved for treatment of psoriasis. Alefacept mediates reduction of circulating memory T cells in patients and mediates Fc receptor (FcR) 2 -dependent cell-mediated killing of T cells in vitro (3, 4). It is hypothesized that alefacept both reduces deleterious effector functions of activated T cells by blocking interaction of CD2 with CD58 and deletes autoaggressive T cells through FcR-dependent killing. When CD16A is the activating FcR each of the individual interactions of alefacept is low affinity with a K d of 1.5 M for the CD2-CD58 interaction and K d of 0.91 M for the Fc-CD16 interaction (5-8). How these solution affinities relate to interactions in an adhesion area is not clear.These interactions are proposed to take place in the twodimensional interface between cells, but there are only limited data on such interactions and no quantitative data on formation of the trimolecular bridges as proposed for alefacept. Determining such parameters in a model system would be a first step to development of a system of two-dimensional pharmacology to better predict in vivo behavior in cell-cell conta...