Enhancing NK Cell Activity in Colorectal Cancer with an Fc-Optimized Antibody Targeting CD276 (B7-H3)

Sylwia A Stefańczyk,1– 3 Xenija Kaiser,1– 3 Ilona Hagelstein,1– 3 Martina S Lutz,1– 3 Samuel J Holzmayer,1– 3 Latifa Zekri,1– 3 Melanie Märklin,1– 3 Susanne Jung1– 4 1Clinical Collaboration Unit Translational Immunology, Department of Internal Medicine, University Hospital of Tübingen, Tübingen, Germany; 2German Cancer Consortium (DKTK), Partner Site Tübingen, Partnership Between DKFZ and University Hospital Tübingen, Tübingen, Germany; 3Cluster of Excellence iFIT (EXC 2180) Image-Guided and Functionally Instructed Tumor Therapies, University of Tübingen, Tübingen, Germany; 4Department of Peptide-Based Immunotherapy, Institute of Immunology, University and University Hospital Tübingen, Tübingen, GermanyCorrespondence: Susanne Jung, Clinical Collaboration Unit Translational Immunology, Department of Internal Medicine, University Hospital of Tübingen, Otfried-Müller-Str 10, Tübingen, 72076, Germany, Tel +4970712983275, Fax +4970712925407, Email [email protected] & Aims: Colorectal cancer (CRC) represents a major global health burden due to its high incidence and mortality, particularly in the advanced stages, with limited treatment options. While therapies that block the PD-1/PD-L1 pathway have demonstrated clinical benefit, their success in CRC is largely confined to patients with high microsatellite instability (MSI-H) tumors. Innovative immunotherapeutic solutions are critically needed for the majority of CRC cases. CD276 (B7-H3), a B7 family immune checkpoint molecule, is overexpressed in many cancers including CRC. Significant CD276 upregulation in CRC cell lines compared to benign tissues has been previously demonstrated. In this study, the potential of an Fc-optimized anti-CD276 monoclonal antibody to enhance natural killer (NK) cell activity in CRC was evaluated.Methods: An Fc-optimized monoclonal antibody, 8H8_SDIE, was developed to enhance NK cell activity by increasing CD16 binding. In vitro experiments were conducted using human CRC cell lines and peripheral blood mononuclear cells (PBMCs) from healthy adult donors to evaluate the binding specificity of 8H8_SDIE to CD276. NK cell activation was assessed by measuring the upregulation of activation markers (CD69, CD25, and CD107a) and secretion of cytotoxic mediators (IFNγ, granzyme B, and perforin). Cytotoxicity assays were performed to determine NK cell-mediated tumor cell lysis.Results: 8H8_SDIE specifically bound to CD276-positive CRC cells and significantly enhanced NK cell activation. These effects included increased levels of activation and cytotoxic mediators. In cytotoxicity assays, 8H8_SDIE demonstrated potent NK cell-mediated lysis of CRC cells.Conclusion: The Fc-optimized anti-CD276 antibody 8H8_SDIE effectively enhanced NK cell reactivity against CD276-positive CRC cells and induced tumor cell lysis in vitro. These findings suggest that 8H8_SDIE holds potential as a novel immunotherapeutic candidate for CRC, particularly for patients with microsatellite-stable disease, and warrants further evaluation in advanced preclinical and future clinical studies.Plain Language Summary: Colorectal cancer (CRC) ranks among the most frequently diagnosed malignancies and remains a major contributor to cancer-related mortality on a global scale. Many patients with advanced CRC do not respond well to current treatments, and only a small number benefit from existing immunotherapies. This highlights the urgency of developing alternative treatments that could benefit a broader range of patients.In this study, we focused on a protein called CD276, which is often found in large amounts in cancer cells, including colorectal cancer cells. We created a specially engineered antibody (a type of immune molecule that helps the body recognize and attack threats) targeting CD276. This antibody, named 8H8_SDIE, was optimized to stimulate natural killer (NK) cells - a type of immune cell capable of eliminating tumor cells. We tested the antibody in the laboratory on colorectal cancer cells. The antibody successfully bound to cancer cells and strongly activated NK cells. After activation, NK cells released cytotoxic molecules which induced cancer cell death.These results suggest that 8H8_SDIE could be a promising new immunotherapy for colorectal cancer. Although more studies are needed before this can be used to treat patients, this study demonstrates a potential approach to improving treatment options for people with this challenging disease.Keywords: B7-H3, Fc engineering, colon cancer, bench to bedside, immunotherapy, ADCC