Endoglin-aptamer-functionalized liposome-equipped pd-1-silenced t cells enhance antitumoral immunotherapeutic effects

Abstract

Background: The broader application of adoptive cell therapy (ACT) in cancer immu-notherapies (particularly for solid tumors) has always been limited by the immunosuppres-sive tumor microenvironment (TME) and the insufficient targetability of effector T cells, resulting in unsatisfied therapeutic outcome. Here, we designed a new strategy by using aptamer-based immunoliposomes to modify PD-1-silencing T cells, which were activated by dendritic cell (DC)/tumor fusion cells (FCs) to improve the antitumor potency of cytotoxic T lymphocytes (CTLs/CD8+ T cells). Methods: PD-1 gene was knocked out from CD8+ T cells using CRISPR/Cas9 system to liberate T cell activity from immunosuppression. The PD-1− T cells were stimulated with DC/tumor FCs, followed by further functional modification of tumor-specific nanoliposomes (hEnd-Apt/CD3-Lipo) to generate FC/PD-1− CTLs. The activation and proliferation and specificity of the modified FC/PD-1− CTLs were measured. The antitumor activity of these CTLs against HepG2-tumors was evaluated in xenograft NOD/SCID mice, and the antitumor mechanism was investigated based on tissue immunohistochemistry and serum ELISA. Results: Our results indicated that the modification of hEnd-Apt/CD3-Lipo nanocomposites on the FC/PD-1− CTLs had a more substantial synergetic effect in inhibiting tumor growth and prolonging animal survival, rather than other control liposomes. Furthermore, the hEnd-Apt/CD3-Lipo-modified FC/PD-1− CTLs showed a stronger antitumor outcome in the tumor-bearing mouse model, through the mechanisms of suppressing tumor cell proliferation, promoting tumor apoptosis, reducing angiogenesis but increasing the infiltration of the FC/ PD-1− CTLs in the tumor tissue, as well as upregulating the systemic levels of IFN-γ, IL-2, TNF-α and IL-6 cytokines, by comparison of the control settings. Conclusion: In sum, our investigation suggests an enhancement of antitumor effect by the surface modification of endoglin-targeting nanoliposomes upon DC/tumor FC-activated PD-1− CTLs, therefore, provides a new tumoral endoglin-targeted approach as a promising strategy to reduce immunosuppression of tumor microenvironment and improve the immu-notherapeutic outcome of anticancer ACT.