Design, engineering, and functional evaluation of nanobody-based anti-CD73 for immunogenic cell death induction in chemoresistant colorectal Cancer cell line.

[BACKGROUND] CD73 is a key immunosuppressive ectoenzyme overexpressed in colorectal cancer (CRC), facilitating tumor immune evasion by generating adenosine. While monoclonal antibodies have shown promise, their limited tissue penetration restricts efficacy. Nanobodies, due to their small size and high stability, provide a novel therapeutic platform. This study aimed to design, produce, and functionally evaluate a recombinant anti-CD73 nanobody-based protein capable of stimulating anti-tumor immunity in a tumor cell line.

[METHODS] We engineered heavy-light nanobody (HeLiNa-73), a novel anti-CD73 nanobody, through CDR grafting from the clinical antibody IPH5301 onto a nanobody scaffold. HeLiNa-73 was expressed in E. coli, purified, and characterized for binding affinity, cytotoxicity, and induction of immunogenic cell death (ICD) markers. Functional assays assessed calreticulin (CALR) exposure, HMGB1 release, ATP secretion, apoptosis, and dendritic cell (DC) maturation.

[RESULTS] HeLiNa-73 exhibited favorable binding to CD73 and potent cytotoxicity in HT-29 CRC cells (IC₅₀ = 23.99 μg/mL). Treatment markedly increased CALR exposure (12.4 % vs. 1.2 % control, p < 0.05) and HMGB1 release (>10-fold, p < 0.001), though ATP release remained unchanged. HeLiNa-73 significantly promoted apoptosis and enhanced DC maturation, with CD80/CD86 upregulation upon coculture with treated tumor cells.

[CONCLUSION] HeLiNa-73 combines CD73 inhibition with ICD induction, thereby overcoming adenosine-mediated immune suppression while enhancing tumor immunogenicity. These in vitro findings highlight HeLiNa-73 as a next-generation nanobody-based candidate for in vivo chemoresistant CRC studies and to synergize with checkpoint blockade.