Development of galectin-1 and galectin-7-specific inhibitors: Immunotherapy and molecular imaging in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, accounting for 12-17% of cases, and is associated with increased metastasis and recurrence. Despite significant advances in recent years, notably the approval of anti-PD-1 immunotherapy as part of the standard treatment for TNBC, current therapeutic options remain limited, are often associated with adverse effects, and are effective for only a minority of patients. Therefore, there is an urgent need to develop new therapeutic strategies. Galectins (GALs) are a family of glycan-binding proteins recognized as glyco-immune checkpoints in cancer. They play key roles in several processes of tumor progression, including immunosuppression, metastatic dissemination, and treatment resistance. In TNBC, GAL-1 and GAL-7 are overexpressed in ~40% of cases and are associated with increased metastasis, poor prognosis, and reduced survival. Despite more than two decades of research, most GAL inhibitors developed have shown limited success, partly due to their lack of specificity. We report the development of novel, specific inhibitors targeting GAL-1 and GAL-7 using camelid antibody-derived nanobodies (Nbs). Lead Nbs were conjugated with the NOTA chelator, labeled with copper-64 ( 64 Cu), and utilized as radiotracers to detect GAL-1 and GAL-7 expression in primary tumors of a TNBC mouse model via positron emission tomography (PET). Additionally, GAL-1 and GAL-7 Nbs were fused to a human IgG1 Fc fragment to generate two novel minibodies (Mbs) homologs, G1M1 and G7M8, to assess the therapeutic potential of galectin inhibition as monotherapies and in combination with anti-PD-1 immunotherapy in a preclinical TNBC model. We identified several high-affinity Nbs specific to GAL-1 and GAL-7, with no detectable cross-reactivity to other galectins. Lead candidates effectively inhibited both GALs binding to T-cell glycoreceptors, preventing T-cell apoptosis in vitro. In PET imaging, 64 Cu-labeled Nbs demonstrated specific and optimal accumulation in TNBC 20 h post-administration. Inhibition of GAL-1 and GAL-7 showed therapeutic potential in a TNBC mouse model, reducing lung metastases and modulating both local and systemic immune responses through distinct mechanisms. Notably, G1M1 demonstrated efficacy comparable to anti-PD1 therapy. In summary, we developed highly specific GAL-1 and GAL-7 inhibitors that not only inhibit T-cell apoptosis but also serve as effective diagnostic and therapeutic agents in TNBC. These findings offer a promising strategy to overcome previous limitations in galectin inhibitor development, laying a foundation for novel therapeutic approaches in cancer treatment and for better understanding the role of galectins in cancer and other diseases.