CD40 激动剂抗体 (αCD40) 在临床前和早期临床研究中均显示出良好的抗肿瘤反应。然而，其全身给药与免疫和肝毒性相关，这阻碍了其临床使用。此外，αCD40 显示出较低的肿瘤保留率并诱导 PD-L1 表达，从而使肿瘤微环境 (TME) 具有免疫抑制作用。为了克服这些问题，在本研究中，我们开发了一种多功能免疫体，其中 αCD40 缀合在表面，而小分子免疫调节剂 RRX-001 封装在其中。免疫体在给药 96 小时前表现出较高的肿瘤积累，并在体内表现出 αCD40 的持续释放。免疫体通过增加 TME 中 CD45 CD8 T 细胞、CD45 CD20 B 细胞、CD45 CD11c DC 和 F4/80 CD86 细胞的数量，显着延迟肿瘤生长，并在患有 GL-261 胶质母细胞瘤的小鼠中显示出无肿瘤存活。免疫体显着减少了 T 调节细胞、M2 巨噬细胞和 MDSC 的数量，并降低了 PD-L1 的表达。此外，与 Th2 细胞因子（IL-4 和 IL-10）相比，免疫体显着提高了 Th1 细胞因子（IFN-γ、IL-6、IL-2）的水平，从而支持抗肿瘤反应。最有趣的是，免疫体通过降低丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、IL-6、IL-1α的水平来避免与游离αCD40相关的体内毒性，并减少肝损伤程度。此外，免疫体治疗的长期存活小鼠表现出肿瘤特异性免疫记忆反应，可在再次攻击时阻止肿瘤生长。我们的结果表明，这种新型制剂可以在临床上进一步探索，以提高 αCD40 的体内抗肿瘤功效，具有持久的肿瘤特异性免疫，同时减少相关毒性。版权所有 © 2024。由 Elsevier Ltd 出版。

CD40 agonist antibodies (αCD40) have shown promising anti-tumor response in both preclinical and early clinical studies. However, its systemic administration is associated with immune- and hepato-toxicities which hampers its clinical usage. In addition, αCD40 showed low tumor retention and induced PD-L1 expression which makes tumor microenvironment (TME) immunosuppressive. To overcome these issues, in this study, we have developed a multifunctional Immunosome where αCD40 is conjugated on the surface and RRX-001, a small molecule immunomodulator was encapsulated inside it. Immunosomes showed higher tumor accumulation till 96 h of administration and displayed sustained release of αCD40 in vivo. Immunosomes significantly delayed tumor growth and showed tumor free survival in mice bearing GL-261 glioblastoma by increasing the population of CD45+CD8+ T cells, CD45+CD20+ B cells, CD45+CD11c+ DCs and F4/80+CD86+ cells in TME. Immunosome significantly reduced the population of T-regulatory cells, M2 macrophage, and MDSCs and lowered the PD-L1 expression. Moreover, Immunosomes significantly enhanced the levels of Th1 cytokines (IFN-γ, IL-6, IL-2) over Th2 cytokines (IL-4 and IL-10) which supported anti-tumor response. Most interestingly, Immunosomes averted the in vivo toxicities associated with free αCD40 by lowering the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-6, IL-1α and reduced the degree of liver damage. In addition, Immunosomes treated long-term surviving mice showed tumor specific immune memory response which prevented tumor growth upon rechallenge. Our results suggested that this novel formulation can be further explored in clinics to improve in vivo anti-tumor efficacy of αCD40 with long-lasting tumor specific immunity while reducing the associated toxicities.Copyright © 2024. Published by Elsevier Ltd.