Anti-IL22 Monoclonal antibody, clone 33VSUJ [PE]

Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N termini of an anti-IL22 antibody, through either the light chain, the heavy chain, or both chains. Although the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay due to the N-terminal degradation. When a glutamine residue was introduced at the N terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody like molecule.

IL-17A is an important cytokine in intestinal inflammation. However, anti-IL-17A therapy does not improve clinical outcomes in patients with Crohn's disease. We aimed to evaluate the role of ROR gamma t(+) innate lymphoid cells (ILCs) in murine colitis models in the absence of IL-17A. An acute colitis model was induced with either dextran sulfate sodium (DSS) or trinitrobenzenesulfonic acid (TNBS) and a chronic colitis model was induced by CD4(+)CD45RB(hi) T cell transfer from either wild-type C57BL/6 or Il17a(-/-) mice. An anti-IL-17A antibody, secukinumab, was also used to inhibit IL-17A function in the colitis model. Flow cytometry was performed to analyze the population of ROR gamma t(+) ILCs in the colonic lamina propria of mice with chronic colitis. Acute intestinal inflammation due to DSS and TNBS was attenuated in IL-17A knockout mice, whereas chronic colitis was not relieved by T cell transfer from Il17a(-/-) mice (% of original body weight: wild-type mice vs. Il17a(-/-) mice, 81.9% vs. 82.2%; P = 0.922). However, the mean proportion of Lin-ROR gamma t(+) lymphocytes was higher after T cell transfer from Il17a(-/-) mice than that after T cell transfer from wild-type mice (28.8% vs. 18.5%). The proportion of Lin-ROR gamma t(+) was also increased in Rag2(-/-) mice that received T cell transfer from wild-type mice when anti-IL-17A antibody was administered (31.7%). Additionally, Il6 and Il22 tended to be highly expressed after T cell transfer from Il17a(-/-) mice. In conclusion, ROR gamma t(+) ILCs may have an important role in the pathogenesis of chronic colitis in the absence of IL-17A. Blocking the function of IL-17A may upregulate Il6 and recruit ROR gamma t(+) ILCs in chronic colitis, thereby upregulating IL-22 and worsening the clinical outcomes of patients with Crohn's disease.