PO.CL05.02 · 临床研究

靶向 uPAR 的 CAR-NK 细胞增强对急性髓系白血病的细胞毒性

uPAR-targeting CAR-NK cells enhance cytotoxicity against acute myeloid leukemia

编号 5199 展板 17 时间 4/21 09:00–12:00 区域 Section 40 主讲 Xingliang Guo, PhD

分会场 Adoptive Cell Therapy 2

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作者与单位 Authors & Affiliations

Xingliang Guo¹, Nadima Uprety², Rejeena Shrestha², Francia Reyes Silva¹, Sunil Acharya², Bijender Kumar³, Bin Liu², Dexing Fang², Bingqian Hu², Sha Wang², May Daher¹, Ana K. Nunez Cortes², Corry Jones², LaTretta Harris², Vernikka Woods², Jerrell E. Scott², April Gilbert², Cornelio Santibanez², Katayoun Rezvani², Rafet Basar¹

¹Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX,²Institute for Cell Therapy Discovery and Innovation, The University of Texas MD Anderson Cancer Center, Houston, TX,³Division of Hematology Oncology, VCU Massey Comprehensive Cancer Center, Richmond, VA

摘要 Abstract

中文摘要

背景：复发/难治性急性髓系白血病（AML）仍然难以治疗，凸显了对新疗法的需求。基于 NK 细胞的免疫疗法提供了强效的抗肿瘤活性，同时 GVHD 或严重 CRS 的风险较低。尿激酶纤溶酶原激活物受体（uPAR），由 PLAUR 编码，在恶性和衰老细胞上高表达，而在健康 HSC 和大多数正常重要器官上基本缺失。我们发现 PLAUR 在 AML 中显著上调，并证实原代原始细胞上 uPAR 表面强表达。基于这一有利特征，我们开发了一个现货型 uPAR 靶向 CAR-NK 平台，带有 CD28 共刺激结构域和分泌型 IL-15 以增强持久性。方法：将脐带血来源的 NK 细胞用 IL-12/15/18 预激活，并转导以表达 (i) uPAR-CAR、(ii) IL-15 或 (iii) uPAR-CAR/IL-15。分别通过流式细胞术、ELISA 和 CyTOF 评估 CAR 表达、细胞因子产生和表型。通过 IncuCyte 实时杀伤和 Annexin V 染色测试对 AML 细胞系（MV4-11、THP-1、OCI-AML3）和原代原始细胞的抗白血病活性。通过序贯肿瘤再攻击评估长期活性。在移植了荧光素酶标记的 OCI-AML3 并接受单次 NK 输注的 NSG 小鼠中评估体内疗效。结果：uPAR 在 AML 原始细胞上高表达，但在健康 HSC/HSPC 上极少，证实了其作为 CAR-NK 靶点的适用性。uPAR-CAR/IL-15 NK 细胞被高效生成，分泌 IL-15，并强力扩增。在体外，与仅 CAR、仅 IL-15 或未转导的 NK 细胞相比，它们对所有 uPAR+ AML 细胞系和原代原始细胞显示出显著增强且抗原特异性的杀伤（p < 0.0001），而 PLAUR 敲除的细胞抵抗杀伤。uPAR-CAR/IL-15 NK 细胞产生更多的 IFN-gamma 和 TNF-alpha，并在反复肿瘤暴露期间维持更优的功能，CyTOF 揭示了一个富含激活/细胞毒性的元簇。在体内，单次 uPAR-CAR/IL-15 NK 输注显著减少了白血病负荷、延缓了进展，并改善了骨髓中的 NK 持久性。与未转导的 NK 相比，外周血（p < 0.05）、骨髓（p = 0.001）和脾脏（p < 0.01）中的肿瘤负荷显著减少，未观察到毒性或体重减轻。综合而言，uPAR 是一个安全、选择性的 AML 靶点。uPAR-CAR NK 细胞，尤其是在装配 IL-15 时，表现出强效、抗原特异性且持久的抗白血病活性，支持推进该平台用于 AML 治疗。

查看英文原文 English abstract

Background: Relapsed/refractory acute myeloid leukemia (AML) remains difficult to treat, highlighting the need for new therapies. NK cell-based immunotherapy offers potent antitumor activity with low risk of GVHD or severe CRS. The urokinase plasminogen activator receptor (uPAR), encoded by PLAUR , is highly expressed on malignant and senescent cells while largely absent from healthy HSCs and most normal vital organs. We found PLAUR significantly upregulated in AML and confirmed strong uPAR surface expression on primary blasts. Based on this favorable profile, we developed an off-the-shelf uPAR-targeted CAR-NK platform with a CD28 costimulatory domain and secreted IL-15 to enhance persistence. Methods: Cord blood-derived NK cells were preactivated with IL-12/15/18 and transduced to express (i) uPAR-CAR, (ii) IL-15, or (iii) uPAR-CAR/IL-15. CAR expression, cytokine production, and phenotype were assessed by flow cytometry, ELISA, and CyTOF, respectively. Antileukemic activity against AML cell lines (MV4-11, THP-1, OCI-AML3) and primary blasts was tested by IncuCyte real-time killing and Annexin V staining. Long-term activity was assessed by sequential tumor rechallenge. In vivo efficacy was evaluated in NSG mice engrafted with luciferase-labeled OCI-AML3 and treated with a single NK infusion. Results: uPAR was highly expressed on AML blasts but minimal on healthy HSCs/HSPCs, confirming its suitability as a CAR-NK target. uPAR-CAR/IL-15 NK cells were efficiently generated, secreted IL-15, and expanded robustly. In vitro, they showed markedly enhanced and antigen-specific killing of all uPAR+ AML lines and primary blasts compared with CAR-only, IL-15-only, or non-transduced NK cells ( p < 0.0001), while PLAUR -knockout cells resisted killing. uPAR-CAR/IL-15 NK cells produced more IFN-gamma and TNF-alpha and maintained superior function during repeated tumor exposure, with CyTOF revealing an activation/cytotoxicity-enriched metacluster. In vivo, a single uPAR-CAR/IL-15 NK infusion significantly reduced leukemia burden, delayed progression, and improved NK persistence in bone marrow. Compared with non-transduced NKs, tumor burden in peripheral blood ( p < 0.05), bone marrow ( p = 0.001), and spleen ( p < 0.01) was substantially reduced, with no observed toxicity or weight loss. Taken together, uPAR is a safe, selective AML target. uPAR-CAR NK cells, particularly when armored with IL-15, display potent, antigen-specific, and durable antileukemic activity, supporting advancement of this platform for AML therapy.

利益披露 Disclosure

X. Guo, None.. R. Shrestha, None.. F. Reyes Silva, None.. S. Acharya, None.. B. Liu, None.. B. Hu, None.. S. Wang, None.. A. K. Nunez Cortes, None.. C. Jones, None.. L. Harris, None.. V. Woods, None.. J. E. Scott, None.. A. Gilbert, None.. C. Santibanez, None.

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