Tinostamustine–nivolumab therapy is associated with antigen-specific cellular and humoral immune responses in heavily pre-treated patients with advanced melanoma

ENIgMA is a phase IB trial (NCT03903458; SNCTP000003243) evaluating the safety and preliminary activity of the first-in-class alkylating deacetylase inhibitor tinostamustine in combination with nivolumab (3 mg/kg) in patients with predominantly immune checkpoint inhibitor (ICI)-resistant, advanced melanoma. We conducted this longitudinal immune monitoring study in 15 of the 17 enrolled patients to characterize systemic immunologic changes associated with an immunomodulatory dose of tinostamustine. We have previously demonstrated that combination of tinostamustine with nivolumab was able to induce disease stabilization in 54% and confirmed partial responses in 23% of evaluable patients. Peripheral blood samples collected longitudinally during treatment were analysed by flow cytometry, serum antigen-specific IgG enzyme-linked immunosorbent assay (ELISA), and intracellular cytokine staining following ex vivo peptide stimulation. Longitudinal flow cytometric analyses demonstrated dynamic changes in peripheral immune subsets, including T cells, natural killer (NK) cells and monocytes, suggesting ongoing immune engagement during tinostamustine–nivolumab therapy. In progressors, elevated baseline lactate dehydrogenase (LDH) levels significantly and inversely correlated with circulating T cell frequencies, indicating distinct immune profiles associated with poor clinical outcome. Antigen-specific adaptive immune responses were detectable despite extensive prior treatment and high tumour burden. CD4+ T cell responses, particularly TNFα-associated responses directed against melanoma-associated antigens, represented the dominant cellular immune activation pattern. Additionally, treatment related increases in melanoma-associated antigen-specific IgG responses were also observed in several patients. Collectively, these findings demonstrate that immunomodulatory doses of tinostamustine in combination with nivolumab are linked with preserved adaptive immune competence and detectable cellular and humoral immune activation in heavily pre-treated patients with advanced melanoma.