PO.CL05.01 · 临床研究
利用脂质纳米颗粒和环状RNA对小细胞肺癌进行DLL3靶向体内CAR-T治疗
DLL3-targeted in vivo CAR-T therapy of small cell lung cancer with lipid nanoparticles and circular RNA
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：小细胞肺癌（SCLC）的生存情况令人沮丧，亟需开发新型靶点和有效的治疗方法。Delta样配体3（DLL3）是SCLC治疗的一个有前景的靶点，目前有多种积极的治疗策略正在推进，包括DLL3靶向嵌合抗原受体T（CAR-T）细胞治疗。在此，我们报告了一种由环状RNA（circRNA）和抗体偶联脂质纳米颗粒（LNP）构建的DLL3靶向体内CAR-T治疗，在治疗SCLC方面展现出有前景的治疗潜力。
方法：环状RNA（circRNA）是共价闭合的单链RNA，能够抵抗外切酶介导的降解。与线性mRNA相比，环状RNA作为翻译模板更为稳定和高效，因此circRNA被认为是mRNA的“下一代”。RNA-LNP通过微流控法将circRNA水溶液与脂质组分乙醇溶液以3:1的比例混合制备而成。LNP通过SATA-马来酰亚胺化学反应与纯化的大鼠抗小鼠CD5抗体（CD5-LNP）或IgG（IgG-LNP）偶联，获得抗体偶联的LNP-circRNA。
结果：我们此前曾报道过一种高效的基于TREM1/DAP12的新型多链CAR结构。在本研究中，我们成功合成并验证了编码DLL3靶向CAR的circRNA的可行性。为量化体内CAR-T细胞的百分比，将LNP静脉注射入8周龄小鼠。注射后24小时通过流式细胞术表征免疫器官和外周血中的CAR-T细胞。与非靶向LNP（未偶联LNP和IgG偶联LNP）相比，CD5偶联的LNP在外周血（约15%）和免疫器官（淋巴结约12%、脾脏约16%、胸腺约22%）中显示出显著更高的CAR-T百分比。我们进一步分析了CAR-T细胞在体内的持续时间。将CD5靶向LNP以每只小鼠30 ug RNA的剂量静脉注射入8周龄小鼠。在每个时间点分离免疫器官和外周血并通过流式细胞术进行表征。我们观察到，表达CAR的T细胞在24小时达到峰值，随后在5天后完全消失。最后，利用小鼠皮下和原位模型，我们证实了基于circRNA的CAR-T治疗在根除SCLC肿瘤方面的疗效，显著延长了生存期。在注射后第28天，实验组中3/4的小鼠仍存活，而对照组的所有小鼠在第21天前均已死亡。各指定组间未见肝毒性（以AST和ALT指示）的显著升高，提示良好的安全性特征。
结论：借助抗体偶联LNP和circRNA，我们有效地在体内构建了DLL3靶向CAR-T细胞，其对SCLC显示出有效的细胞毒性。本研究为推进SCLC治疗和体内CAR-T治疗提供了一条有前景的途径。
查看英文原文 English abstract
Background: The survival of small cell lung cancer (SCLC) is discouraging, necessitating the development of novel targets and effective therapeutic approaches. Delta-Like Ligand 3 (DLL3) represents a promising target for the treatment of SCLC, with ongoing vigorous therapeutic strategies, including DLL3-targeted chimeric antigen receptor-T (CAR-T) cell therapy. Here, we report DLL3-targeted in vivo CAR-T therapy constructed by circular RNA (circRNA) and antibody-conjugated lipid nanoparticles (LNP), yielding promising therapeutic potential for treating SCLC.
Methods: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs that are resistant to exonuclease-mediated degradation. Compared with linear mRNA, circular RNA is more stable and efficient as translate templates, thus circRNA has been considered as “the next generation” of mRNA. RNA-LNPs were prepared via microfluidic mixing an aqueous solution of the circRNA and an ethanolic solution of the lipid components at a ratio of 3:1. LNPs were conjugated with purified rat anti-mouse CD5 (CD5-LNP) or IgG (IgG-LNP) via SATA-maleimide chemistry reaction to obtain antibody conjugated LNP-circRNA.
Results: We have previously reported a highly efficient TREM1/DAP12-based novel multiple-chain CAR structure. In current study, we successfully synthesized and validated the feasibility of circRNA encoding DLL3-targeted CAR. To quantify the percentage of CAR-T cell in vivo, LNPs were intravenously injected into 8-week-old mice. CAR T cells in the immune organ and peripheral blood were characterised via flow cytometry 24h post injection. CD5-conjugated LNP showed a significant higher CAR-T percentage in peripheral blood (~15%) and immune organs (~12% in lymph node, ~16% in spleen, and 22% in thymus) compared to non-targeted LNP (unconjugated LNP and IgG-conjugated LNP). We further analysed the CAR-T cell duration in vivo. CD5-targeted LNP were intravenously injected into 8-week-old mice at dose of 30 ug RNA per mice. Immune organ and peripheral blood were isolated and characterised via flow cytometry at each time point. We observed that the CAR expressing T cells reached peak at 24h, then completely diminished after 5days. Lastly, using mice subcutaneous and orthotopic models, we demonstrated the efficacy of circRNA-based CAR-T therapy in SCLC tumor eradication, significantly extending survival. By Day 28 post-injection, 3/4 mice in the experimental group remained alive, whereas all in the control group had perished by Day 21. No significant increase of liver toxicity (indicated by AST and ALT) among the indicated groups, suggested favorable safety profile.
Conclusion: Leveraging antibody-conjugated LNP and circRNA, we have effectively engineered DLL3-targeted CAR-T cells in vivo, which shows effective cytotoxicity against SCLC. This research offers a promising avenue for advancing SCLC and in vivo CAR-T therapeutics.
利益披露 Disclosure
Y. Ma, None..
J. Cai, None..
Y. Xia, None..
X. Huang, None..
M. Qiu, None.