Antigen-scaffolds loaded with hyper-stable neoleukin-2/15 expand antigen-specific T cells with a favorable phenotype for adoptive cell therapy.

INTRODUCTION

Adoptive cell therapy (ACT) has shown promising results in cancer treatment, however, achieving effective ex vivo expansion of potent, functionally active, and cytotoxic T cells remains challenging. To address this challenge, we recently developed artificial antigen-presenting scaffolds (Ag-scaffolds) capable of expanding antigen-specific T cells with phenotypes favorable for ACT. Here, we compared the established technology using IL2/IL21-loaded Ag-scaffolds (Ag-IL2/21) with scaffolds incorporating Neoleukin-2/15 (Ag-Neo2/15), an engineered cytokine that selectively signals via IL-2Rβ/γ complexes to enhance CD8+ T cell proliferation while limiting regulatory T cell expansion.

METHODS

Antigen-specific T cells were expanded ex vivo using Ag-IL2/21 or Ag-Neo2/15 scaffolds. Expansion efficiency, cytokine production, and cytotoxic activity were assessed. Functional and transcriptional states were profiled using single-cell sequencing, including cytotoxic and dysfunction gene signature scoring. T cell receptor (TCR) sequencing was performed to evaluate clonal expansion.

RESULTS

Ag-Neo2/15 scaffolds supported robust expansion of antigen-specific CD8+ T cells at levels comparable to Ag-IL2/21 scaffolds. Ag-Neo2/15-expanded CD8+ T cells exhibited increased effector cytokine production and durable cytotoxicity against tumor cells. Single-cell transcriptomic analysis revealed higher cytotoxic gene signature scores, reduced dysfunctional scores, and increased expression of genes linked to increased proliferation in Ag-Neo2/15-expanded T cells. TCR sequencing demonstrated preferential expansion of CD8+ T cell clones enriched for transcriptional features associated with antigen-experienced effector states and sustained effector function.

DISCUSSION: Ag-Neo2/15 scaffolds enhance the quality of ex vivo-expanded antigen-specific T cells by promoting a cytotoxic, less dysfunctional, and highly proliferative phenotype. These findings identify Ag-Neo2/15 as a promising improvement for ACT manufacturing, optimizing the balance between expansion capacity and durable effector function, with potential implications for improving cancer immunotherapy outcomes.