PO.CL06.01 · 临床研究
神经节苷脂靶向：对神经母细胞瘤的甜蜜复仇
Ganglioside targeting: Exerting sweet revenge on neuroblastoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
神经母细胞瘤（NBL）是儿童中最常见的诊断出的颅外实体瘤，导致11%的儿童癌症死亡。Dinutuximab是一种FDA批准的抗GD2单克隆抗体（mAb），用于治疗高危NBL。尽管将dinutuximab纳入了一线治疗方案，但仍有40-50%的患者复发并最终死于该疾病。对抗GD2治疗耐药的机制知之甚少。此前，我们已经证明异种移植物在对抗GD2免疫治疗的反应中出现GD2丢失。这种耐药机制是由一种酶ST8SIA1的下调驱动的，而GD2通路中的其他酶保持不变。我们假设ST8SIA1的丢失导致GD2减少和GM2的代偿性增加。在本研究中，我们验证了我们的假设，将GM2作为免疫治疗靶点进行审查，为此我们设计了GM2特异性嵌合抗原受体（CAR）和mAb，并在鼠模型中测试了它们的疗效和安全性。首先，我们在3个转移性NBL异种移植模型中测试了抗GM2 mAb作为单一疗法。无论初始疗效如何，单一疗法未能提供持久反应，最终所有小鼠均复发。然而，由于每种抗体在GD2高表达模型中均显示出部分活性，我们在下一个实验中增加了一个联合治疗组。联合治疗被证明更为优越，因为所有接受该治疗的小鼠均实现了肿瘤根除和长期生存。随后，我们测试了基于用于mAb的同一结合物的抗GM2 CAR-T细胞，观察到强劲而持久的反应。使用串联GM2-GD2 CAR双重靶向GD2和GM2，能够完全消除肿瘤并提供长期生存。神经节苷脂在正常组织中的分布知之甚少。然而，已知GD2存在于周围神经中，在那里引起神经性疼痛，即主要的剂量限制性毒性。为了预判该联合疗法可能带来的潜在毒性，我们优化了冰冻组织的IHC神经节苷脂检测，并成功筛查了健康组织微阵列（TMA）。GM2显示出有限的组织分布，仅限于肾脏和甲状腺。更重要的是，未观察到GD2和GM2之间的重叠。我们还筛查了鼠组织以寻找安全性模型。C57Bl/6小鼠的肾脏显示出与人肾脏相当的GM2水平，因此被用于毒性研究。GM2 mAb耐受性良好，没有脱靶毒性的证据，无体重减轻或明显的病理特征。最后，承蒙NANT提供，我们获取了此前接受抗GD2治疗的复发NBL患者的活性冰冻骨髓样本。这些样本显示出异质性的GD2，但GM2较高。综上，这些数据表明免疫治疗驱动的GD2丢失伴随着GM2的同时增加。更重要的是，它验证了GM2作为GD2低表达NBL的替代靶点，并且由于GM2的正常组织表达有限，可以与GD2一起安全地靶向以防止抗原逃逸。
查看英文原文 English abstract
Neuroblastoma (NBL) is the most common extracranial solid tumor diagnosed in children and is responsible for 11% of pediatric cancer deaths. Dinutuximab is an FDA approved monoclonal antibody (mAb) against GD2, used in the treatment of high-risk NBL. Despite integration of dinutuximab into upfront treatment protocols, 40-50% of patients still relapse and eventually die of their disease. Resistance to anti-GD2 therapy is poorly understood. Previously, we have shown GD2 loss in xenografts in response to anti-GD2 immunotherapy. This resistance mechanism is driven by downregulation of one enzyme, ST8SIA1 while other enzymes in the GD2 pathway remain unchanged. We hypothesized that loss of ST8SIA1 leads to reduced GD2 and a compensatory increase in GM2. In the present work, we validate our hypothesis, vetting GM2 as an immunotherapy target for which we engineered GM2-specific chimeric antigen receptors (CAR) and mAbs and tested their efficacy and safety in murine models.First, we tested anti-GM2 mAbs as a monotherapy in 3 metastatic NBL xenograft models. Regardless of initial efficacy, monotherapy fail to provide long-lasting responses and eventually all mice relapsed. However, because each antibody showed partial activity in the GD2-high model, we added a combination-therapy arm in the next experiment. The combination proved to be superior, as all mice receiving it saw tumor eradication and long-term survival.We then tested anti-GM2 CAR T cells, based on the same binder used for mAbs, and saw robust and durable responses. Dual targeting GD2 and GM2, with a tandem GM2-GD2 CAR, was able to eliminate the tumors completely and provided long-term survival.Ganglioside distribution in normal tissue is poorly understood. However, it is known that GD2 is present in peripheral nerves, where it causes neuropathic pain, the main dose-limiting toxicity. To anticipate potential toxicities the combination could present, we optimized ganglioside detection by IHC on frozen tissue and successfully screened healthy tissue microarrays (TMAs). GM2 showed limited tissue distribution, being restricted to the kidneys and thyroid. More importantly, no overlap between GD2 and GM2 was observed.We also screened murine tissues from in search of a safety model. The kidneys of C57Bl/6 mice showed GM2 levels comparable to human kidneys, so were used for toxicity studies. GM2 mAbs were well tolerated with no evidence of off-target toxicity, no weight loss or noticeable pathological features.Finally, courtesy of NANT, we procured viably frozen bone marrow samples from relapsed NBL patients, previously treated with anti-GD2. These samples showed heterogeneous GD2, yet high GM2.Together these data shows, GD2 loss driven by immunotherapy and a concomitant increase in GM2. More importantly, it validates GM2 as an alternative target for GD2 low NBL and because GM2 has limited normal-tissue expression, it can be safely targeted alongside GD2 to prevent antigen escape
利益披露 Disclosure
G. N. Dalton, None..
W. Kim, None..
K. H. Lu, None..
N. W. Mabe, None..
M. Rotiroti, None..
S. Ghazarian, None..
A. Marachelian, None.