Background: The pleiotropic cytokine macrophage migration inhibitory factor (MIF) is associated with tumor aggressiveness, metastasis, and disease progression. Due to its ubiquitous nature, MIF is considered an unsuitable target for therapeutic intervention. Oxidized MIF (oxMIF) is the disease-related, tumor-specific isoform of MIF, which undergoes a structural change enabling selective oxMIF-targeting by antibodies. We now developed ON203, a bioengineered anti-oxMIF antibody with highly improved biophysiochemical and biological properties, designed to exert better tumor retention and effector functions. Material and Methods: After physicochemical characterization of the optimized anti-oxMIF antibody ON203, cell-based assays were performed to evaluate antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Tumor penetration was assessed by infrared-labeled ON203 injected in tumor-bearing mice. Human cancer cell-line (PC3) xenografted mice and human colorectal carcinoma 3D tumoroids were treated with ON203, and efficacy was assessed either by tumor volume measurements and immunohistochemical analysis or by tumor-cell killing and immune cell composition assessment. Results: ON203 demonstrated strong immune cell effector functions in reporter- and PBMC-mediated tumor cell killing assays. In xenograft models, ON203’s accumulation and retention in the tumor tissue was proven and tumor volumes were significantly reduced upon treatment. Tumor cell proliferation (assessed by Ki67 staining quantification) and tumor vessel density (CD31 staining quantification) were strongly decreased in the ON203-treated tumors compared to the isotype control. Tumor-infiltrating immune cells provided further insights on the immunomodulatory therapeutic effects of ON203. Strikingly, freshly isolated tumoroids from colorectal carcinoma patients responded not only with tumor cell death, but a clear immunomodulatory effect on the tumor microenvironment in responding tumoroids was detected: ON203 activated NK and NKT cells and supported M1 polarization. Conclusions: Our results demonstrate direct antitumorigenic effects of the anti-oxMIF antibody ON203 (i) by blocking the biologic function of oxMIF thereby reducing tumor cell proliferation and angiogenesis and (ii) by immunomodulation of the tumor microenvironment. In the upcoming Phase 1 trial we will evaluate ON203’s safety, tolerability, pharmacokinetics, and pharmacodynamics in patients with solid tumors. ON203 bears a high potential as a standalone therapy or in rational combinations with immune checkpoint inhibitors or antiangiogenic agents in the treatment of solid tumors. Conflict of interest: Ownership: Alexander Schinagl, Michael Thiele