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. Citation Format: Yishan Ma, Jingsheng Cai, Yufei Xia, Xiaonan Huang, Mantang Qiu. DLL3-targeted in vivo CAR-T therapy of small cell lung cancer with lipid nanoparticles and circular RNA abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3717.
Ma et al. (Fri,) studied this question.