AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes

Abstract

Affinity of integrin lymphocyte function-associated antigen 1 (LFA-1) is enhanced by conformational changes from the low-affinity closed form to the high-affinity (HA) open form of the ligand-binding inserted (I) domain as shown by work with purified I domains. However, affinity up-regulation of LFA-1 on the cell surface by physiological agonists such as chemokines has yet to be demonstrated by monovalent reagents. We characterize a mAb, AL-57 (activated LFA-1 clone 57), that has been developed by phage display that selectively targets the HA open conformation of the LFA-1 I domain. AL-57 discriminates among low-affinity, intermediate-affinity, and HA states of LFA-1. Furthermore, AL-57 functions as a ligand mimetic that binds only upon activation and requires Mg2+ for binding. Compared with the natural ligand intercellular adhesion molecule-1, AL-57 shows a tighter binding to the open I domain and a 250-fold slower off rate. Monovalent Fab AL-57 demonstrates affinity increases on a subset (approximately 10%) of lymphocyte cell surface LFA-1 molecules upon stimulation with CXCL-12 (CXC chemokine ligand 12). Affinity up-regulation correlates with global conformational changes of LFA-1 to the extended form. Affinity increase stimulated by CXCL-12 is transient and peaks 2 to 5 min after stimulation.

Fig. 1.

SPR analysis of binding by the α_L I domains to Abs AL-57 and MHM24. The HA (K287C/K294C), IA (L161C/F299C), or low-affinity WT I domain was perfused onto immobilized Abs in the presence of 1 mM MgCl₂ (A) or 10 mM EDTA (B). The concentration series for MHM24 was 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 nM. For AL-57, the concentration series was 15.6, 31.3, 62.5, 125, and 250 nM for the HA I domain and 31.25, 62.5, 125, 250, and 500 nM for WT and IA I domains. In all cases, higher concentrations gave higher responses (except that differences are not visible for WT with AL-57 in Mg²⁺ and for HA with AL-57 in EDTA).

Fig. 2.

Acidic residue D101 of AL-57 is critical for binding. (A) Primary sequence of variable regions of AL-57. The CDRs are shown in bold and are underlined. The beginning of a constant region is underlined with a dashed line. (B) Binding of parental and mutant AL-57 IgG (3 μg/ml) to the isolated, locked HA I domain expressed on K562 cells was examined by immunofluorescent flow cytometry in the presence of 1 mM Mg²⁺ (filled histograms) or 5 mM EDTA (open histograms). Binding of AL-57^D101A in Mg²⁺ and EDTA is hardly distinguishable from background binding of control IgG.

Fig. 3.

Binding of AL-57 IgG to WT LFA-1 activated by agonists as determined by immunofluorescent flow cytometry. K562 transfectants expressing WT LFA-1 and IL-15-cultured human primary T lymphocytes were stained for 20 min at 37°C with 20 μg/ml AL-57, TS2/4, or isotype-matched human or mouse control IgG in Hepes-buffered saline containing 1 mM MgCl₂/1 mM CaCl₂, 5 mM MgCl₂/1 mM EGTA, or 1 mM MnCl₂. Cells were washed and stained with FITC-conjugated goat anti-human or anti-mouse Abs. Staining with AL-57 and TS2/4 is shown as solid lines, and background staining with control IgGs is shown as dashed lines. Numbers within the panels show the specific mean fluorescence intensity of AL-57 and TS2/4.

Fig. 4.

The kinetics of LFA-1 activation after stimulation with CXCL-12. Representative histograms for a time course of AL-57 epitope expression are shown. T lymphocytes were stimulated with 50 ng/ml CXCL-12 for the indicated time period. Cells were stained with Fab AL-57 or control Fab labeled with Alexa 488 for 2 min by adding Abs 2 min before the end of stimulation. Cells were then fixed and subjected to flow cytometry. Background staining with control Fab is shown in the open histograms. Numbers within the panels show specific mean fluorescence intensity values for Fab AL-57. At 0, 10, and 20 min, differences between AL-57 Fab and control Fab binding are hardly visible.