Prostate cancer (PC) is a major disease affecting men's health and is the second most frequently diagnosed cancer in men worldwide. Although most newly diagnosed cases of PC are well-differentiated tumors with a high probability of cure, some patients develop aggressive malignancies with an increased risk of recurrence and metastasis. CD276 is overexpressed in many tumors and is often associated with poor prognosis. It is also classified as a B7/CD28 family member, functioning as an antigen-independent co-stimulatory ligand with immunomodulatory properties. Recently, extracellular vesicles (EVs) have been recognized as key players in intercellular communication, transporting membrane proteins, lipids, RNAs, cytosolic proteins, and other signaling molecules to target cells. Furthermore, natural killer (NK) cells offer several advantages over T cells, including greater tumor-killing capacity and a safer profile for allogeneic transplantation. The present study aimed to develop CD276-targeting extracellular vesicles loaded with cisplatin, designed to trigger NK-mediated cytolysis for the treatment of advanced prostate cancer.

Recombinant anti-human CD276 and CD16 proteins, linked to CD81 and CD63 exosomal markers, were derived from genetically engineered and transiently transfected human 293T cells. The expression of CD276 in prostate cancer cells was evaluated using flow cytometry. NK cells were expanded and induced from peripheral blood mononuclear cells (PBMCs) using OKT-3 (anti-CD3) recombinant protein in combination with specific cytokines.

Analysis from the DriverDBv4 database revealed that CD276 expression was significantly higher in primary solid tumor tissues than in normal tissues (p = 0.00069). Additionally, CD276 was highly expressed in prostate PC-3 cancer cells. After two weeks of induction, approximately 90% of the expanded cells were CD3⁻/CD56⁺. Moreover, CD276- and CD16-secreting HEK293T cells were successfully sorted for further expansion and extracellular vesicle production.

These findings suggest that CD276-targeting extracellular vesicles were successfully constructed and could facilitate NK cell infiltration. Furthermore, prostate PC-3 cancer cells, which exhibit high CD276 expression, provide an appropriate model for evaluating the therapeutic efficacy of CD276-targeting EVs loaded with chemotherapeutics. Additionally, CD16-containing EVs may enhance NK cell-mediated cytolysis.