Abstract
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 two-dimensional 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/microm(2) and two-dimensional association constants of 3.1 and 630 microm(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.
Highlights
Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc
CD16B and r1 is the surface concentration of Alefacept bound to CD16B but not bound to CD2; b1 and b2 are the surface concentrations of bridging bonds composed of alefacept with one or both CD58 sites bound to the T cell and their Fc site bound to the CD16B on the bilayer
Where b1 and b2 are the surface concentration of alefacept with one or both CD58s bound to CD2 on the T cell and its Fc bound to Fc receptor (FcR) on the planar bilayer. e1 and e2 are the surface concentration of alefecept with one or both CD58 sites bound to CD2 and its Fc portion free. r is the surface concentrations of free CD16B and r1 is the surface concentration of Alefacept bound to CD16B but not bound to CD2. rT is the surface concentration of FcR on bilayer prior to cell interaction
Summary
FcR, Fc receptor; PBL, human peripheral blood lymphocytes; FITC, fluorescein isothiocyanate; GPI, glycophosphatidylinositol; MR, maximal release; SR, spontaneous release; GFP, green fluorescent protein; BF, bright field; IRM, interference reflection microscopy; D, diffusion coefficient. The interactions of CD2 with CD58 has been studied in detail using supported planar bilayers and the Golan-Zhu analysis revealing a two-dimensional Kd in the range of 1.1–7.6 molecules/m2 and a maximal binding (Bmax) close to the total number of cell surface CD2 accessible by antibodies [6, 12] These results are consistent with self-assembly of an ordered interface into which CD2 diffuses freely. A key prediction of the model is that the concentration of alefacept needed to initiate an adhesion area is inversely proportion to the square of the CD2 density This prediction is important because it implies that a 5-fold higher expression of a receptor on a target compared with a bystander cell will result in initiation of interaction with the target at 25-fold lower concentration of alefacept than needed to initiate adhesion to the bystander. The data indicate that rules governing access of alefacept-CD2 complexes to the adhesion area are not fully understood and identify areas for further study
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