PO.TB05.02 · 肿瘤生物学
在免疫功能健全的MYCN驱动小鼠神经母细胞瘤同种异体移植模型中评价TCR模拟型CAR T细胞的临床前治疗
Evaluating TCR mimetic CAR T cell preclinical therapeutics in an immunocompetent MYCN-driven murine neuroblastoma allograft model
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：可操作的免疫功能健全小鼠模型可填补过继细胞疗法临床前评价中的一个关键空白。以肽为中心（PC）的嵌合抗原受体（CAR）T细胞靶向由HLA-A*24:02（A24）呈递的、源自神经母细胞瘤胞内癌蛋白PHOX2B的9聚体肽的pMHC，目前已进入针对复发性神经母细胞瘤的1期临床试验（NCT07007117）。为研究治疗局限性和增强策略，我们开发了一种靶向PHOX2B/A24的小鼠（m）PC-CAR同源模型。
方法：将C57BL/6（BL/6）可穿透的TH-MYCN来源的同种异体移植物和细胞系用嵌合人/鼠MHC HLA-A*24:02/H-2Kb进行改造，以呈递保守的PHOX2B 9聚体肽。使用编码第二代mPC-CAR（含临床scFv连接鼠CD28或4-1BB及CD3ζ）的MSCV逆转录病毒载体来创建稳定的GPE86生产者细胞系。纳入截短的mCD19或荧光素酶以监测转导效率和体内迁移。将BL/6脾细胞在体外用人（h）IL-2激活并转导，并在hIL-7/15中扩增。使用流式细胞术、ELISA、实时细胞阻抗和O-link蛋白质组学进行多重功能测定。
结果：转导效率可重复地介于40-60%之间。mPC-CAR-T细胞在14天内扩增15至20倍，同时维持记忆和效应免疫表型的平衡，制造后耗竭标志物极少。针对表达PHOX2B-A24-H-2Kb的细胞系，含CD28ζ或4-1BBζ共刺激域的mPC-CAR-T细胞表现出强劲的IFN-gamma、IL-2和TNFalpha分泌，以及强效的细胞毒性，效应细胞:靶细胞比率低至0.5:1，在制造后长达14天及冷冻保存后仍然有效。CD28ζ的mPC-CAR T细胞在连续肿瘤再攻击时维持100%的细胞毒性，而4-1BBζ的细胞毒性在3次攻击后限于50%。在无外源性细胞因子支持的情况下，肿瘤暴露诱导了CD44+IL7Ralpha-终末效应表型以及耗竭标志物CD39、CTLA-4和Tim-3的上调。评估抗肿瘤效力、持久性和疗效障碍的体内研究正在进行中，将另行报告。
结论：我们在BL/6小鼠中创建了一个可操作的MHC杂交TH-MYCN来源的神经母细胞瘤同种异体移植模型。该模型目前正被用于研究多种PC-CAR增强策略，如mRNA编码表位疫苗接种和细胞因子武装，并配合空间转录组学，这将为我们靶向PHOX2B的PC-CAR T细胞的进一步临床开发提供依据。
查看英文原文 English abstract
Background: Tractable immunocompetent murine models for adoptive cellular therapies could fill a critical gap in preclinical evaluations. Peptide-centric (PC) chimeric antigen receptor (CAR) T cells targeting pMHC of a 9mer peptide derived from the neuroblastoma intracellular oncoprotein PHOX2B presented by HLA-A*24:02 (A24) is now in Phase 1 clinical trial for relapsed neuroblastoma (NCT07007117). To study therapeutic limitations and enhancement strategies, we developed a syngeneic model of PHOX2B/A24-targeting murine (m)PC-CARs.
Methods: C57BL/6 (BL/6)-penetrant TH-MYCN-derived allografts and cell lines were engineered with a chimeric human/murine MHC HLA-A*24:02/H-2K b to present conserved PHOX2B 9mer peptide. MSCV retroviral vectors encoding second-generation mPC-CARs containing the clinical scFv conjoined to murine CD28 or 4-1BB with CD3ζ were used to create stable GPE86 producer cell lines. Truncated mCD19 or luciferase was included to monitor transduction efficiency and in vivo trafficking. BL/6 splenocytes were activated and transduced ex vivo with human (h)IL-2, and expanded in hIL-7/15. Multiplex functional assays were performed using flow cytometry, ELISA, real-time cell impedance, and O-link proteomics.
Results: Transduction efficiency reproducibly ranged from 40-60%. mPC-CAR-T cells expanded 15 to 20-fold over 14 days while maintaining balanced memory and effector immunophenotypes with minimal exhaustion markers post-manufacture. Against PHOX2B-A24-H-2K b -expressing cell lines, mPC-CAR-T cells with CD28ζ or 4-1BBζ costimulatory domains exhibited robust IFN-gamma, IL-2, and TNFalpha secretion and potent cytotoxicity down to an effector:target ratio of 0.5:1 up to 14 days post-manufacture and after cryopreservation. CD28ζ mPC-CAR T cells maintained 100% cytotoxicity upon serial tumor rechallenge, whereas cytotoxicity of 4-1BBζ was limited to 50% after 3 challenges. In the absence of exogenous cytokine support, tumor exposure induced CD44 + IL7Ralpha - terminal effector phenotype and upregulation of exhaustion markers CD39, CTLA-4, and Tim-3. In vivo studies to assess anti-tumor potency, persistence, and barriers to efficacy are ongoing and will be reported.
Conclusion: We created a tractable MHC hybrid TH-MYCN-derived neuroblastoma allograft model in BL/6 mice. This model is currently being deployed to study various PC-CAR enhancement strategies such as mRNA encoded epitope vaccination and cytokine armoring with accompanying spatial transcriptomics that will inform further clinical development of our PHOX2B-directed PC-CAR T cells.
利益披露 Disclosure
E. Posthill, None..
M. Samanta, None..
D. Groff, None..
C. Casey, None..
T. Acholla, None..
A. Giudice, None..
K. R. Bosse, None..
R. Wang, None..
T. T. Spear, None..
J. M. Maris, None.