Engineering a Ph-Sensitive Humanized Infliximab with Improved Potency and Developability Using STEM™

Abstract Background Tumor necrosis factor-α inhibitors such as infliximab and adalimumab have transformed autoimmune disease treatment; however, infliximab is a mouse-human chimeric antibody that remains immunogenic, is associated with self-association/aggregation liability, and requires prolonged intravenous administration. We humanized infliximab and engineered infliximab-derived candidates with improved potency and developability. Methods Infliximab complementarity-determining regions were grafted onto human germline frameworks to generate humanized infliximab. STage-Enhanced Maturation (STEM TM ) technology produced an affinity-matured clone (hInBG4), followed by targeted amino-acid substitutions in the complementarity-determining regions to generate LW2Y, LW2YR2S, and LW2YHR1K. Variants were evaluated by a cell-based tumor necrosis factor alpha neutralization assay, affinity-capture self-interaction nanoparticle spectroscopy, a baculovirus particle enzyme-linked immunosorbent assay, size-exclusion high-performance liquid chromatography, transient expression in human embryonic kidney 293 cells, and tumor necrosis factor alpha binding kinetics by biolayer interferometry, including dissociation at pH 7.4 and 5.8. Results All three variants showed two-to three-fold higher neutralization potency than chimeric infliximab and outperformed adalimumab. Affinity-capture self-interaction nanoparticle spectroscopy shifts decreased from double-digit parental values to low single digits, while baculovirus particle binding ratios remained acceptable. Size-exclusion chromatography showed cleaner monomer peaks with reduced tailing, and expression increased relative to humanized infliximab. LW2Y combined very high affinity at pH 7.4 with markedly faster dissociation at pH 5.8, consistent with pH-dependent antigen release. Conclusions Humanization, affinity maturation, and targeted complementarity-determining region re-engineering generated infliximab-derived candidates with improved potency and developability and identified LW2Y as a lead for further preclinical evaluation. Statement of Significance STEM™ engineering produced a pH-sensitive, humanized infliximab lead with improved potency and developability. LW2Y shows two-to three-fold stronger TNF-α neutralization, low self-association, improved expression, and accelerated antigen dissociation at pH 5.8, supporting advancement into preclinical pharmacokinetic and efficacy studies.