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

基于嵌合抗原受体(CAR)的细胞免疫治疗扩大了侵袭性B细胞淋巴瘤和急性淋巴细胞性白血病的治疗范围，自体CAR-T产品现已常规应用于临床。然而，产生自体细胞产品的重大后勤障碍使这种治疗方式变得繁琐，并且仅适用于选定的一组患者。异基因脐带血来源的自然杀伤(NK)细胞有望克服这些障碍，作为一种现成的产品，可以在不需要完全配型的情况下使用。多发性骨髓瘤是一种不可治愈的克隆性浆细胞恶性肿瘤，尽管采用了蛋白酶体抑制剂和免疫调节药物等新物质，但总生存期中位数为6年。尽管BCMA指导的自体CAR-T疗法的前景有望进一步改善临床结果，但由于BCMA阴性疾病的出现和体内持续时间有限而导致的最终复发仍然是一个挑战。多特异性靶向，其中CAR修饰的免疫细胞被设计成识别多种肿瘤相关抗原，有望克服抗原丢失导致的免疫逃逸。天然配体CARS依赖于生理上发生的高亲和力配体选择性地与其受体结合，从而允许同时靶向多个肿瘤相关抗原。在多发性骨髓瘤中，APRIL是一种天然的配体，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型的双特异三聚体APRIL-CAR-NK结构，它使用多顺反子逆转录病毒载体平台复制APRIL的自然三聚体构象，以增强浆细胞结合亲和力。我们将使用基于流式细胞术的细胞毒性分析来研究特定的浆细胞溶解，并通过共聚焦显微镜观察免疫突触的形成来增强。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特征并提高CAR-NK细胞的适合性。免疫反应在很大程度上依赖于细胞内的营养水平，尤其是MYC信号是NK细胞代谢结构的关键调节因子。因此，我们假设，调节MYC信号的活性可以使CAR-NK细胞在代谢上更加健壮，并增强体内的持久性。为了解决这个问题，我们将针对他们的翻译后调控网络，特别是相扑-泛素串扰，来微调细胞内MYC的水平。重新编程的CAR-NK细胞将使用基于质谱学的代谢组学和单细胞转录图谱的多OMICS方法来深入表征，以阐明它们改变的代谢组学特征。在功能上，重新编程的CAR-NK细胞将使用BCMA阴性的多发性骨髓瘤小鼠模型在体内进行验证。

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.