The project successfully implemented a comprehensive set of in vitro and in vivo studies to characterize the immunological effects of MDC-735. In vitro co-culture experiments demonstrated that MDC-735 induces robust pro-inflammatory responses, including elevated levels of IL-6 and TNF-α, and significantly enhances T-cell activation as measured by CD69 expression. These effects were specific to the macrophage–drug conjugate and were not observed with the free drug or alternative protein–drug complexes, highlighting the functional superiority of MDC-735.
In immunocompetent orthotopic glioma mouse models, MDC-735 treatment resulted in profound remodeling of the tumor microenvironment. Treated tumors exhibited increased infiltration of CD4⁺ and CD8⁺ T cells, B cells, and neutrophils, alongside a marked reduction in T-cell exhaustion markers such as PD-1, TIM-3, and TOX. Importantly, MDC-735 reduced immunosuppressive regulatory T-cell populations in tumor-draining lymph nodes, while sparing non-tumor-associated lymphoid tissues, indicating a localized and tumor-specific immune modulation.
The project further demonstrated that MDC-735 alters the phenotype of administered macrophages toward a pro-inflammatory, antigen-presenting state, characterized by reduced expression of immunosuppressive markers and increased MHC-II expression. Functional validation in immunodeficient mouse models confirmed that the curative effect of MDC-735 critically depends on intact adaptive immunity, particularly CD4⁺ and CD8⁺ T cells.
Collectively, these activities resulted in a comprehensive mechanistic dataset demonstrating that MDC-735 converts an immunosuppressive glioblastoma microenvironment into an immunologically active one and induces a lymphocyte-dependent anti-tumor response.
