Enhancement of antitumor immunity by CTLA-4 antisense oligonucleotides with CpG motif in mice.

Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a critical immune checkpoint molecule that negatively regulates T-cell activation and immune responses. Antibodies blocking CTLA-4 enhance anti-tumor T-cell responses, making them effective cancer immunotherapeutics. However, the CTLA-4 antibodies can trigger significant immune-related adverse events (irAEs). These toxicities may arise in part from antibody-induced complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC) toward normal cells. Here, we report the development of a non-antibody immune checkpoint inhibitor comprising a CTLA-4-targeting antisense oligonucleotide (ASO) incorporating CpG motifs (CpG-CTLA-4 ASO), designated as ISA2. Similar to CpG ODN, a toll-like receptor 9 (TLR9) agonist, ISA2 upregulates costimulatory molecules in both B cells and CD11c+ cells. Simultaneously, ISA2 significantly downregulates CTLA-4 expression in both CD4+ and CD8+ T cell populations. The dual immunomodulatory functions of CpG-CTLA-4 ASO-simultaneously providing costimulatory activation and CTLA-4 suppression-demonstrated remarkable therapeutic efficacy in murine melanoma and colon carcinoma models. The treatment outcomes included significant tumor growth inhibition, prolonged survival of tumor-bearing hosts and achievement of complete tumor regression in a subset of animals. The ISA2 represents a novel class of dual-functional immunotherapy with promising clinical applications. Unlike conventional CTLA-4 antibodies, this agent combines TLR9 agonism with CTLA-4 silencing in a single molecular entity, enabling simultaneous immune activation and checkpoint blockade. This innovative design offers several potential clinical advantages: (1) augmentation of tumor-specific T-cell responses through synergistic modulation of both innate and adaptive immunity; (2) potentially reduced antibody-dependent toxicity compared to conventional antibody-based CTLA-4 blockade therapies; and (3) enhanced delivery flexibility attributable to its favorable small-molecule properties.