Metabolic reprogramming of Trimeric APRIL-CAR-engineered Natural Killer (NK) cells to enhance tumor-cell recognition, in vivo persistence and anti-tumor potency in Multiple myeloma

三聚体 APRIL-CAR 工程自然杀伤 (NK) 细胞的代谢重编程可增强多发性骨髓瘤中的肿瘤细胞识别、体内持久性和抗肿瘤效力

• 批准号: 464778766
• 负责人: Dr. Alexander Biederstädt
• 金额: --
• 依托单位: MD Anderson Cancer Center
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/464778766?language=en
• 关键词: Metabolic; reprogramming; Trimeric; APRIL; CAR

Chimeric-antigen receptor (CAR)-based cellular immunotherapy has augmented the treatment armamentarium for aggressive B cell lymphomas and acute lymphoid leukemia with autologous CAR-T products now routinely applied in the clinic. However, significant logistic obstacles of generating autologous cell products make this treatment modality cumbersome and available only to a selected group of patients. Allogeneic cord blood derived natural killer (NK) cells promise to overcome these hurdles and can be administered without the need for full HLA matching as an off-the-shelve product.Multiple myeloma is an incurable clonal plasma cell malignancy with a median overall survival of 6 years despite adoption of novel substances such as proteasome inhibitors and immunomodulatory drugs. While the outlook of BCMA-directed autologous CAR-T therapies promises to further improve clinical outcomes, eventual relapse due to the emergence of BCMA-negative disease and limited in vivo persistence remain a challenge.Multi-specific targeting, in which CAR-modified immune cells are engineered to recognize multiple tumor-associated antigens, promises to overcome antigen-loss driven immune escape. Natural ligand CARs which rely on physiologically occurring high affinity ligands to selectively bind to their receptor counterparts allow to simultaneously target multiple tumor-associated antigens. In multiple myeloma, APRIL is such a natural ligand and enables CAR-NK cells to engage with the plasma cell specific antigens TACI and BCMA. In the proposed project we plan to engineer a novel dual-specific trimeric APRIL-CAR-NK construct which replicates APRIL's natural trimeric conformation using a multicistronic retroviral vector platform to enhance plasma cell binding affinity. Specific plasma cell lysis will be investigated using flow-cytometry based cytotoxicity assays and augmented by visualization of immunological synapse formation by confocal microscopy.Addressing the limited in vivo persistence of current generation CAR-T therapies, we plan to modulate the immunometabolomic signature and increase CAR-NK cellular fitness. Immune responses are crucially dependent on intracellular nutrient levels and MYC signaling in particular is as a key regulator of NK cell metabolic configuration. We therefore hypothesize that modulating the activity of MYC signaling can render CAR-NK cells more metabolically robust and enhance in vivo persistence. To address this idea, we will fine-tune intracellular MYC levels by targeting their post-translational regulatory networks, specifically the SUMO-Ubiquitin crosstalk. Reprogrammed CAR-NK cells will be characterized in-depth using a multi-OMICS approach relying on mass-spectrometry-based metabolomics and single cell transcriptomic profiling to elucidate their altered metabolomic signatures. Functionally, reprogrammed CAR-NK cells will be validated in vivo using a BCMA-negative multiple myeloma mouse model.

基于嵌合抗原受体（CAR）的细胞免疫疗法已经增强了侵袭性B细胞淋巴瘤和急性淋巴细胞白血病的治疗手段，目前临床上常规应用的自体CAR-T产品。然而，产生自体细胞产品的显著物流障碍使得这种治疗方式繁琐并且仅对选定的患者组可用。同种异体脐带血来源的自然杀伤（NK）细胞有望克服这些障碍，并可以管理，而不需要完全HLA匹配作为一个现成的product.Multiple骨髓瘤是一种不可治愈的克隆浆细胞恶性肿瘤的中位总生存期为6年，尽管采用新的物质，如蛋白酶体抑制剂和免疫调节药物。虽然BCMA导向的自体CAR-T疗法的前景有望进一步改善临床结果，但由于BCMA阴性疾病的出现和有限的体内持久性导致的最终复发仍然是一个挑战。多特异性靶向，其中CAR修饰的免疫细胞被工程化以识别多种肿瘤相关抗原，有望克服抗原丢失驱动的免疫逃逸。天然配体汽车依赖于生理上存在的高亲和力配体来选择性地结合其受体对应物，允许同时靶向多种肿瘤相关抗原。在多发性骨髓瘤中，APRIL是这样一种天然配体，使CAR-NK细胞能够与浆细胞特异性抗原TACI和BCMA结合。在拟议的项目中，我们计划设计一种新的双特异性三聚体APRIL-CAR-NK构建体，其使用多顺反子逆转录病毒载体平台复制APRIL的天然三聚体构象，以增强浆细胞结合亲和力。将使用基于流式细胞术的细胞毒性测定研究特异性浆细胞溶解，并通过共聚焦显微镜观察免疫突触形成来增强。针对当前一代CAR-T疗法在体内的有限持久性，我们计划调节免疫代谢组学特征并增加CAR-NK细胞适应性。免疫应答至关重要地依赖于细胞内营养水平，特别是MYC信号传导作为NK细胞代谢构型的关键调节剂。因此，我们假设调节MYC信号传导的活性可以使CAR-NK细胞在代谢上更稳健，并增强体内持久性。为了解决这个问题，我们将通过靶向其翻译后调控网络，特别是SUMO-泛素串扰来微调细胞内MYC水平。重编程的CAR-NK细胞将使用多OMICS方法进行深入表征，该方法依赖于基于质谱的代谢组学和单细胞转录组学分析，以阐明其改变的代谢组学特征。在功能上，将使用BCMA阴性多发性骨髓瘤小鼠模型在体内验证重编程的CAR-NK细胞。