A Dual-Payload Antibody-Drug Conjugate Targeting CD276/B7-H3 Elicits Cytotoxicity and Immune Activation in Triple-Negative Breast Cancer
Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease that often relapses following treatment with standard radiotherapies and cytotoxic chemotherapies. Combination therapies have potential for treating refractory metastatic TNBC. Here, we aimed to develop an antibody-drug conjugate with dual payloads (DualADC) as a chemo-immunotherapy for TNBC. The overexpression of an immune checkpoint transmembrane CD276 (also known as B7-H3) was associated with angiogenesis, metastasis, and immune tolerance, in over 60% of TNBC patients. Development of a monoclonal antibody (mAb) capable of targeting the extracellular domain of surface CD276 enabled delivery of payloads to tumors, and a platform was established for concurrent conjugation of a traditional cytotoxic payload and an immunoregulating toll-like receptor 7/8 agonist to the CD276 mAb. The DualADC effectively killed multiple TNBC subtypes, significantly enhanced immune functions in the tumor microenvironment, and reduced tumor burden by up to 90-100% in animal studies. Single-cell RNA-sequencing, multiplex cytokine analysis, and histology elucidated the impact of treatment on tumor cells and the immune landscape. This study suggests that the developed DualADC could represent a promising targeted chemo-immunotherapy for TNBC. Overall design: The flash-frozen tissue samples (25 mg) were collected and stored at -80°C until processing. Chromium Next GEM RNA Profiling Sample Fixation Kit (PN-1000414, 10x Genomics, Pleasanton, CA, USA) was used to fix tissue by mixing 1 mL of fixation buffer with 25 mg of tissue, followed by fine mince and incubation at 4°C for 16-24 hours without agitation. After fixation, tissue samples were centrifuged, washed with chilled PBS, and resuspended in 1 mL of tissue resuspension buffer. The fixed tissues supplemented with pre-warmed dissociation buffer were dissociated using an Octo Dissociator according to the manufacturer's instructions. The dissociated tissue samples were filtered through a 30 µm filter, centrifuged and resuspended in 1 mL of chilled quenching buffer. Cell concentration was determined using an automated cell counter with fluorescent nucleic acid staining. The dissociated tumor samples were stored in 50% glycerol with enhancer at -80°C until single-cell library generation.