PO.CL05.08 · 临床研究
胶原结合型IL-12重塑免疫抑制性微环境并改善复发性胶质母细胞瘤的生存
Collagen-binding IL-12 remodels immunosuppresive microenvironment and improves survival in recurrent glioblastoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
复发性GBM（rGBM）是最具侵袭性的恶性脑肿瘤，中位生存期仅为6个月。rGBM构建了严重的免疫抑制性肿瘤微环境（TME），伴有Tregs增多和CD8 T细胞募集受损，使得在其他癌症中有效的免疫疗法失效。虽然IL-12是一种强效的免疫刺激性细胞因子，能够重塑这种免疫抑制性TME，但全身性给药会引起严重毒性，阻碍其临床应用。因此，亟需能够将IL-12选择性递送至rGBM肿瘤的技术。rGBM通过构建独特的细胞外基质（ECM）获得治疗抵抗；因此，我们设想利用这一特性作为储库来富集IL-12。通过筛选，我们聚焦于COL I和III，它们在正常脑组织中表达极低，但在放疗后及rGBM中显著上调。我们通过将IL-12与血管性血友病因子（vWF）的胶原结合结构域（CBD）融合，构建了CBD-IL-12，利用vWF在血管破坏、炎症和肿瘤部位选择性结合COL I和III而不结合正常器官的特性。小鼠rGBM模型中的生物分布实验表明，CBD-IL-12选择性地在rGBM肿瘤中富集，而在其他COL I和III高表达的器官中不富集。小鼠和犬的毒性研究显示，与IL-12相比，CBD-IL-12显著降低了外周IFN-gamma水平，且无肝或胰腺功能障碍。光谱流式细胞术和单细胞RNA测序显示，CBD-IL-12增加了效应记忆CD8 T细胞、M1样巨噬细胞和DCs，同时减少了Tregs、耗竭T细胞、M2样巨噬细胞和MDSCs，显著逆转了免疫抑制。CBD-IL-12（25μg静脉注射，两次）使rGBM小鼠模型的生存期延长了260%，完全缓解（CR）率为13%。CBD-IL-12联合放疗实现了63%的CR率。值得注意的是，在我们的rGBM模型中，单独放疗未带来生存获益，而与CBD-IL-12联合则实现了显著的肿瘤抑制，这可能是通过放疗诱导的COL I和III上调以及增强的巨噬细胞/小胶质细胞募集实现的。长期生存者通过再攻击试验显示出持久的免疫记忆。为实现临床转化，我们构建了人源化CBD-IL-12，并在3D人GBM切片培养中验证了其疗效，证实其增加了CD8 T细胞并减少了Tregs。胶原结合型IL-12克服了rGBM的免疫抑制，具有强效的抗肿瘤作用，代表了一种有前景的新型免疫治疗方法。
查看英文原文 English abstract
Recurrent GBM (rGBM) is the most aggressive malignant brain tumors, with a median survival of 6 months. rGBM establishes a severe immunosuppressive tumor microenvironment (TME) with increased Tregs and impaired CD8 T cell recruitment, rendering immunotherapies effective in other cancers ineffective. While IL-12 is a potent immunostimulatory cytokine capable of remodeling this immunosuppressive TME, systemic delivery causes severe toxicity, hindering clinical application. Therefore, technology to selectively deliver IL-12 to rGBM tumors is critically needed. rGBM acquires treatment resistance through construction of a unique extracellular matrix (ECM); therefore, we conceived of utilizing this as a reservoir to accumulate IL-12. Through screening, we focused on COL I & III, minimally expressed in normal brain but highly upregulated after radiotherapy and in rGBM. We created CBD-IL-12 by fusing IL-12 with the collagen-binding domain (CBD) of von Willebrand factor (vWF), leveraging vWF's selective binding to COL I & III at sites of vascular disruption, inflammation, and tumors while sparing normal organs. Biodistribution assays in a mouse rGBM model demonstrated selective CBD-IL-12 accumulation in rGBM tumors without accumulation in other COL I & III-high organs. Toxicity studies in mice and dogs showed CBD-IL-12 significantly reduced peripheral IFN-gamma levels compared to IL-12 without hepatic or pancreatic dysfunction. Spectral flow cytometry and single-cell RNA-seq revealed that CBD-IL-12 increased effector memory CD8 T cells, M1-like macrophages, and DCs while reducing Tregs, exhausted T cells, M2-like macrophages, and MDSCs, dramatically reversing immunosuppression. CBD-IL-12 (25μg i.v., twice) prolonged survival by 260% with a 13% CR rate in rGBM mice models. Combining CBD-IL-12 with radiotherapy achieved 63% CR. Notably, in our rGBM model where radiotherapy alone provided no survival benefit, combination with CBD-IL-12 achieved dramatic tumor suppression, likely through radiation-induced COL I & III upregulation and enhanced macrophage/microglia recruitment. Long-term survivors demonstrated durable immune memory by rechallenge testing. For clinical translation, we engineered humanized CBD-IL-12 and validated its efficacy in 3D human GBM slice cultures, demonstrating increased CD8 T cells and reduced Tregs. Collagen-binding IL-12 overcomed rGBM immunosuppression with potent antitumor effects, representing a promising novel immunotherapeutic approach.
利益披露 Disclosure
O. Iwaloye, None..
J. Takei, None..
L. Barr, None..
K. Sasaki, None..
H. Ichie, None..
S. Tsuzuki, None..
J. Ishihara, None..
S. Osuka, None.