Abstract 4788: Discovery of HX035, an OX40-bi-epitope bi-specific antibody (BsAb), for the treatment of inflammatory/autoimmune diseases

Abstract With hyperactive OX40+CD4+ T-cells implicated as the pathogenic cells in various inflammatory/autoimmune disorders, we hypothesized that OX40-antagonistic and/or depleting antibody are both important candidate therapeutics for these diseases. To test this hypothesis, we designed and tested a novel bi-specific antibody (BsAb), HX035 and a 1+1 asymmetric IgG1 mAb, against two different OX40 epitopes. Epitope-1 is featured with non-ligand blocking but strong ADCC (depleting antibody), while epitope-2 is characterized by ligand blocking and antagonistic activity. We found that HX035 binds to recombinant OX40 proteins with the similar binding affinity as the respective parental mAbs, with EC50 0.056 nM (avidity) as opposed to 0.027 nM (epitope 1-mAb) and 0.036 nM (epitope 2-mAb), respectively. Although the binding avidity of HX035 to OX40+ cells is similar to its parental mAbs, HX035 showed more binding to OX40lo and even more to OX40hi cells, as anticipated. HX035 is also an OX40 ligand blocker slightly less potency than the epitope 2-mAb (IC50 5.0 nM vs. 2.6 nM). HX035 also showed similar extent of antagonistic activity to epitope2-mAb in an in vitro reporter bioassay. Remarkably, HX035 induced significantly enhanced ADCC over both parental mAbs, making it a stronger depleting antibody than both parental mAbs. We have also further engineered an ADCC-enhanced (5 times stronger) HX035 molecule, so making it an even stronger depleting-antibody. A human PBMC acute graft-versus-host disease (aGVHD) mouse model indeed confirmed the superior anti-aGVHD activities of HX035, over both parental mAbs in the preclinical setting, accompanied with significant depletion of CD4+ T-cells in vivo. All these suggested that HX035 may be a potential “best-in-class” (FIC) candidate for the treatment of autoimmune diseases. Citation Format: Hang Ke, Tao Yang, Feiyu Peng, Jialing Li, Cen Chen, Peixuan Song, Lei Zhang, Faming Zhang, Henry Qixiang Li. Discovery of HX035, an OX40-bi-epitope bi-specific antibody (BsAb), for the treatment of inflammatory/autoimmune diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4788.