RESUMEN
Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.
Asunto(s)
Antiinflamatorios , Citocinas , Citocinas/metabolismo , Inmunidad Innata , Inmunomodulación , Ingeniería de ProteínasRESUMEN
AIM: Bridging immunoassays for the detection of antidrug antibodies (ADAs) are limited to detection of bivalent molecules and are prone to interference by drug and soluble targets. Hence, alternative approaches for ADA detection are desired. Materials & methods: A novel ADA assay with secondary Fc detection using human soluble Fcγ receptor I (hsFcγRI) was established and compared with standard bridging assay. RESULTS: Both assays showed consistent results in human and cynomolgus monkey samples. In contrast to the bridging assay, the hsFcγRI-based assay was insensitive to the presence of oligomeric targets and appeared to have better drug tolerance. CONCLUSION: The hsFcγRI-based ADA assay can serve as alternative screening assay or as orthogonal confirmation method for preclinical and clinical immunogenicity testing of IgG therapeutics lacking Fc effector functions.