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To the Editor: The CD40-CD40 ligand (CD40L, also known as CD154) pathway has been identified as a potential target for pharmaceutical intervention in type 1 diabetes [1, 2]. The CD40-targeted peptide approach published by Wagner and colleagues in Diabetologia in 2014 [3] captured our attention because their data indicated that the peptide used in their research induced potent diabetes prevention and possible diabetes reversal in the gold-standard NOD mouse model of type 1 diabetes. Moreover, in contrast to other anti-CD40L antibodies, it was suggested that the peptide format used in the paper by Wagner’s group [3] would not cause anti-CD40L immune complexes to trigger Fcγ type 2 receptor A (FcγRIIa) signalling through an Fc moiety, thus avoiding platelet activation and the subsequent thrombotic complications associated with these antibodies [4]. The in vivo data obtained byWagner’s group [3] imply that the CD40L-derived 15-mer peptide would bind to CD40 with high enough affinity to block endogenous CD40L binding or to induce any other sort of uncharacterised tolerogenic signalling downstream of CD40. To assess 15-mer peptide binding to CD40, we first produced the mouse 15-mer peptide and a scrambled 15-mer version, confirmed solubility by visual inspection and verified identity by mass spectroscopy (MS; Fig. 1a) and purity via C18 reversed phase HPLC (data not shown) (see ESM Methods for further details). We proceeded to characterise the peptide’s binding association and dissociation kinetics to mouse CD40 via biolayer interferometry using an Octet Red instrument (Fremont, CA, USA) (Fig. 1b). The commercial control mouse CD40L protein (R&D Systems; Minneapolis, MN, USA) demonstrated strong affinity for mouse CD40, estimated in the low nmol/l range, with fast on-rate and slow off-rate binding kinetics. In contrast, very fast on and fast off kinetics were observed for both the 15-mer peptide and the scrambled version of the peptide, possibly indicating nonspecific binding. To evaluate binding using an alternative method, surface plasmon resonance (SPR) was employed to quantitatively determine the affinity of 15-mer binding to mouse CD40-Fc (see ESM Methods). The commercial CD40L protein bound to CD40 with a binding affinity (KD) of 0.4 nmol/l (Fig. 1c), but no binding was seen using up to 3 μmol/l of the 15-mer (Fig. 1d) or scrambled peptides (data not shown). Next, we performed structural analysis by aligning the peptide with the published CD40-CD40L crystal structure in silico in order to evaluate binding location and theoretical interaction quality (see ESM Methods) [5]. This analysis was performed under the assumption that synthesised, free and flexible 15-mer peptide will retain the same conformation as native CD40L when interacting with CD40. It was determined that peptide interaction with CD40 is mediated by residues K143, G144, Y145 and Y146 (hydrophilic contacts), and also E142 and K143 (charge interactions) [5]. George Jeffrey categorised H bonds with donor-acceptor distances of 3.2–4.0 Å as weak and electrostatic; the 15-mer peptide CD40 interaction region was predicted to have H bonds that fell within these donor-accepted distances (see Fig. 1e), [6]. * Ken Coppieters [email protected]