Single-step grafting of aminooxy-peptides to hyaluronan: A simple approach to multifunctional therapeutics for experimental autoimmune encephalomyelitis

Abstract

The immune response to antigens is directed in part by the presence or absence of costimulatory signals. The ability to coincidently present both antigen and, for example, a peptide that inhibits or activates the costimulatory pathway, would be a valuable tool for tolerization or immunization, respectively. A simple reaction scheme utilizing oxime chemistry was identified as a means to efficiently conjugate different peptide species to hyaluronan. Peptides synthesized with an aminooxy N-terminus reacted directly to hyaluronan under slightly acidic aqueous conditions without the need for a catalyst. The resulting oxime bond was found to rapidly hydrolyze at pH 2 releasing peptide, but was stable at higher pH values (5.5 and 7). Two different peptide species, a multiple sclerosis antigen (PLP) and an ICAM-1 ligand (LABL) known to block immune cell stimulation, were functionalized with the aminooxy end group. These peptides showed similar reactivity to hyaluronan and were conjugated in an equimolar ratio. The resulting hyaluronan with grafted PLP and LABL significantly inhibited disease in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Aminooxy-peptides facilitate simple synthesis of multifunctional hyaluronan graft polymers, thus enabling novel approaches to antigen-specific immune modulation.

Co-Delivery of Autoantigen and B7 Pathway Modulators Suppresses Experimental Autoimmune Encephalomyelitis

“…The amount of PLP and context signalbinding peptides conjugated to HA for each of the SAgAs was determined using digestion in acidic medium (4 h at pH 2.0) and then subsequently analyzed by HPLC as previously reported (24). For analysis of SAgA PLP:sF2 , following peptide cleavage, the solution was centrifuged for 5 min at 13,000 rpm and the aggregates were dissolved in DMSO before running both the pellet and supernatant on HPLC.…”

“…SAgAs targeting the B7 pathway were created by co-grafting to hyaluronic acid polymers (HA) both autoantigen (PLP 139-151 ) and B7-binding peptides (B7AP, CD80-CAP, or sF2) as previously reported (15,24,25). For a positive control, an ICAM-1-targeted SAgA (SAgA PLP:LABL ) was created containing autoantigen (PLP [139][140][141][142][143][144][145][146][147][148][149][150][151] ) and the cell adhesion peptide LABL derived from leukocyte function associated antigen-1 (LFA-1), which has previously shown efficacy in EAE (24,25).…”

“…The creation of an antigen-specific treatment capable of suppressing the immune response by co-delivery of autoantigen and immune inhibitor may improve the safety and efficacy of autoimmune therapies. Several previous studies have shown that co-delivering autoantigen and immune inhibitory peptide can inhibit autoimmune disease in animal models (22)(23)(24)(25)(26)(27)(28).…”

“…We grafted to hyaluronic acid both a myelin sheath antigenic peptide (PLP [139][140][141][142][143][144][145][146][147][148][149][150][151] ) and peptides capable of binding B7 signaling pathway surface receptors by following previously reported procedures (15,24,25). The resulting soluble antigen arrays (SAgAs) act as a carrier to co-deliver multiple copies of the autoantigen and a peptide from the B7 pathway.…”

“…Conjugation of Aminooxy Peptides to HA Polymer. Singlestep grafting of aminooxy peptides to 16 kDa HA was performed as described previously (24). Briefly, HA was dissolved in 20 mM acetate-buffered solution (pH 5.5±0.1) and each aminooxy reactive peptide was added simultaneously.…”