Inhibition of CD40–CD154 costimulatory pathway by a cyclic peptide targeting CD154

Abstract

Disruption of the CD40-CD154 interaction was found to be effective in the prevention and treatment of several immune-mediated diseases. The antibody-based strategy of inhibition was in humans limited by platelet activation leading to thrombotic effects. Other strategies different from antibody technology may be useful to create tools to interfere with CD40-CD154 pathway. In the present study, we selected and characterized from a phage display library, cyclic hepta-peptides specific for human CD154 through biopanning against plate-immobilized recombinant hCD154-muCD8. Nine phage clones were selected for the ability to bind CD154 expressed on the surface of J558L cells transfected with human CD154. From the nine selected phage clones, we obtained seven different amino acidic sequences, and the corresponding hepta-peptides rendered cyclic by two cysteines were synthesized. All the peptides specifically bound CD154 expressed on J558L. However, only the peptide 4.10 (CLPTRHMAC) was found to recognize the active binding site of CD154, as it competed with the blocking anti-CD154 antibody. When changes in the amino acid composition were introduced in the sequence of 4.10 peptide, the binding to CD154 was abrogated, suggesting that the amino acid sequence was critical for its specificity. This peptide was found to inhibit the CD40-CD154 interaction, preventing CD40-dependent activation of B lymphocytes in vitro as it was able, as the blocking anti-human CD154 mAb, to prevent the expression of CD80 and CD86 costimulatory molecules and switching of Ig isotype induced by CD154. Moreover, the peptide 4.10 inhibited the in vitro endothelial cell motility and organization into capillary-like structures, and the in vivo angiogenesis of human umbilical cord-derived endothelial cells implanted in Matrigel in severe combined immunodeficiency mice. In vitro studies on platelet activation demonstrated that the 4.10 peptide, at variance of the anti-CD154 mAb, was unable to prime human platelet activation and aggregation. In conclusion, we identify a cyclic hepta-peptide able to displace the binding of human CD154 to CD40 expressed on cell surface and to abrogate some biological effects related to the CD40 stimulation, such as B cell activation and endothelial triggered angiogenesis.

Organic dyes as small molecule protein–protein interaction inhibitors for the CD40–CD154 costimulatory interaction

“…The interaction of CD154 on T cells and its receptor CD40 on B cells is essential for lymphocyte signaling leading to T cell-dependent B cell proliferation, immunoglobulin class switching, and B cell maturation. Peptides interfering with the CD40-CD154 interaction have been reported recently in the literature with activities ranging from quite high (IC 50 s of 50-100 nM for relatively large cyclic structures, MW % 2500, with trimeric symmetry (Fournel et al, 2005)) through medium (IC 50 s of 100 mM for two end-group blocked peptides (Allen et al, 2005) and $50 mM for a cyclic heptapeptide (Deambrosis et al, 2009)) to very low (IC 50 > 1 mM for three recombinant phage proteins (Kitagawa et al, 2005)). A series of dipyridine derivatives were claimed in a patent (Zheng et al, 2007), but this is a first report of a series of active small molecules (some with MW < 600) with adequate CD40-CD154 inhibitory activity confirmed in cell assays and with evidence for a degree of selectivity toward this particular PPI.…”

Syndecan-1 promotes the angiogenic phenotype of multiple myeloma endothelial cells

“…Primary cultures of human umbilical vein ECs (HUVECs) were isolated as described previously. 32 Cell types were maintained in culture with endothelial basal medium (EBM), completed with human epidermal growth factor, hydrocortisone and bovine brain extract (all from Cambrex Bioscience, Walkersville, MD, USA), with 10% fetal bovine serum (FBS).…”

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

“…Peptide inhibitors have been explored as possible alternatives. For example, a CD40–CD40L blocking cyclic heptapetide (CLPTRHMAC) has been shown not to prime human platelet activation and aggregation in in vitro platelet activation studies, contrary to the anti-CD40L mAb also tested 39 . More recently, a CD40-targeting peptide (VLQWAKKGYYTMKSN designated as mouse KGYY 15 ), which was designed from the CD40L domain critical for interaction with CD40, has been shown to be effective in preventing T1D in NOD (non-obese diabetic) mice, a well-known animal model of this disease 19 .…”