PO.IM01.07 · 免疫学

靶向CD123和CD33的记忆样NK细胞可增强对急性髓系白血病的杀伤

Memory-like NK cell targeting to CD123 and CD33 improves killing of acute myeloid leukemia

编号 135 展板 9 时间 4/19 02:00–05:00 区域 Section 7 主讲 Emily Phillips, BS;MD

分会场 Alternative Cell Type and in Situ Cell Therapies

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作者与单位 Authors & Affiliations

Emily Phillips¹, Lyra Morina¹, Lynne Marsala¹, Wilbur Song¹, Michelle Becker-Hapak¹, Yoojin Ahn¹, Sushanth Pureti¹, Elizabeth Juarez Diaz¹, Sadia Afrin¹, Pamela Wong¹, Jennifer Tran¹, Joseph Rueve¹, Allison Burdi¹, Nancy Marin¹, Melissa Marie Berrien-Elliott¹, Martin Felices², Jeffrey S. Miller³, Todd A. Fehniger¹

¹Washington University School of Medicine in St. Louis, St. Louis, MO,²Masonic Cancer Center, Minneapolis, MN,³University of Minnesota, Minneapolis, MN

摘要 Abstract

中文摘要

背景：记忆样自然杀伤（ML NK）细胞是一类NK细胞，在受到IL12/15/18刺激后经历分化过程，发展出更好的代谢适应性并增强对多种恶性细胞的杀伤能力。它们是针对急性髓系白血病（AML）的一种有前景的细胞治疗手段，而对于经历AML复发的儿童和成人患者，其预后仍然很差。ML NK细胞过继治疗在成人和儿童患者中的I期研究显示出令人鼓舞的结果，若干患者达到完全缓解，但许多患者在数月后仍会复发。我们研究了两种通过将记忆样NK细胞靶向AML表面抗原CD33和CD123来改善其对AML识别能力的策略。目的：我们通过工程化改造使ML NK细胞表达嵌合抗原受体（CD123 CAR ML NK），从而将ML NK细胞靶向CD123。我们通过一种三特异性杀伤衔接器（CD33 TriKE）将ML NK细胞靶向CD33，该衔接器通过结合ML NK细胞上的CD16和AML上的CD33来增强免疫突触。CD33 TriKE还含有IL-15，以增强ML NK的活化和存活。我们还探索了这两种技术是否能够协同发挥作用以防止免疫逃逸。方法：在一系列短期和长期实验中，比较了使用ML NK细胞与CD123 CAR ML NK细胞、ML NK细胞加CD33 TriKE或二者联合治疗AML的效果。AML靶细胞包括MOLM13、THP-1和原代AML母细胞。在6小时共孵育中评估干扰素γ的产生。使用基于流式的杀伤实验在4小时评估体外杀伤，使用基于荧光素酶的实验在24小时评估。使用Incucyte（活细胞成像）在5天内评估肿瘤控制。使用NSG小鼠的THP-1异种移植模型评估体内肿瘤控制和存活。使用NSG-S小鼠的原代AML异种移植模型正在建立中。结果：两种靶向策略均表现出抗原特异性的杀伤反应。与ML NK细胞相比，两种策略均显著增加干扰素γ的产生（CAR vs MOLM13：+16.57%，p=0.0002，原代AML #34：IFNg +9.1% p=0.0092；CD33 TriKE vs MOLM13：+24.42%，p=.0015，AML34：+9.1% p=0.0092）。两种策略在不同E:T比值下也均表现出增强的AML杀伤（CAR vs MOLM13：p=.0011；AML34：p=.0006；CD33 TriKE vs MOLM13：p<.0001；AML34 p=.0026）。CD123 CAR和CD33 TriKE均改善了体内对THP-1的控制（第23天的生物发光——CD123 CAR：p=0.004，CD33 TriKE：p<.0001）。两种疗法均将中位生存期改善了约3周（CD123 CAR：p<.0001，CD33 TriKE：p=.0001）。结论：CD123 CAR ML NK细胞和经CD33 TriKE处理的ML NK细胞均能增强ML NK细胞在体外和体内杀伤AML的能力，并延长荷THP-1小鼠的存活时间。用CD33 TriKE处理CD123 CAR ML NK细胞可能防止抗原逃逸。这两种靶向策略中的任何一种都极易转化到临床环境。

查看英文原文 English abstract

Background: Memory-like natural killer (ML NK) cells are NK cells which upon stimulation with IL12/15/18 undergo a differentiation process to develop improved metabolic fitness and enhanced killing capabilities of different types of malignant cells. They are a promising cellular therapeutic against acute myeloid leukemia (AML), for which the prognosis remains dismal for both pediatric and adult patients who experience relapse of their AML. Phase I studies of adoptive ML NK cell therapy in adult and pediatric patients showed promising results with several patients achieving complete responses, but many go on to relapse after a few months. We investigate two strategies to improve recognition of AML by memory like NK cells by targeting them to AML surface antigens CD33 and CD123. Objective : We target ML NK cells to CD123 by engineering ML NK cells to express a chimeric antigen receptor (CD123 CAR ML NK). We target ML NK cells to CD33 via a trispecific killer engager (CD33 TriKE) that enhances the immune synapse by engaging CD16 on the ML NK cell and CD33 on AML. The CD33 TriKE also contains IL-15 to enhance ML NK activation and survival. We also explore whether both technologies function synergistically to prevent immune escape. Methods : Treatment of AML with ML NK cells vs CD123 CAR ML NK cells, ML NK cells plus CD33 TriKE, or the combination, was compared in series of short and long-term assays. AML targets include MOLM13, THP-1, and Primary AML blasts. Interferon gamma production was assessed in a 6-hour co-incubation. In-vitro killing was assessed at 4-hours using a flow-based killing assay, and at 24 hours using a luciferase-based assay. Tumor control was assessed over 5 days using Incucyte (live cell imaging). Tumor control and survival in vivo was assessed using an THP-1 Xenograft model in NSG mice. Primary AML xenografts using NSG-S mice is under development. Results: Both targeting strategies demonstrate an antigen-specific killing response. Both strategies significantly increase interferon gamma production compared to ML NK cells (CAR vs MOLM13: +16.57%, p=0.0002, Primary AML #34: IFNg +9.1% p=0.0092; CD33 TriKE vs MOLM13: +24.42%, p=.0015, AML34: +9.1% p=0.0092). Both strategies also demonstrated increased AML killing at various E:T ratios (CAR vs MOLM13: p=.0011; AML34: p=.0006; CD33 TriKE vs MOLM13: p<.0001; AML34 p=.0026). Both CD123 CAR and CD33 TriKE improved THP-1 control in vivo (bioluminescence at 23 days-CD123 CAR: p=0.004, CD33 TriKE: p<.0001). Both therapies improved median survival by about 3 weeks (CD123 CAR: p<.0001, CD33 TriKE: p=.0001). Conclusion: CD123 CAR ML NK cells and ML NK cells treated with CD33 TriKE both enhance the abilities of ML NK cells to kill AML in vitro and in vivo , and prolong time of survival of THP-1 bearing mice. Treatment of CD123 CAR ML NK cells with CD33 TriKE may prevent antigen escape. Either of these targeting strategies are highly translatable to the clinical setting.

利益披露 Disclosure

E. Phillips, None.. L. Morina, None.. L. Marsala, None.. W. Song, None.. M. Becker-Hapak, None.. Y. Ahn, None.. S. Pureti, None.. E. Juarez Diaz, None.. S. Afrin, None.. P. Wong, None.. J. Tran, None.. J. Rueve, None.. A. Burdi, None.. N. Marin, None.

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