Investigating anti-Glypican 2 (GPC2) chimeric antigen receptor (CAR) T-cell trafficking and tumor microenvironment (TME) interaction in fully immunocompetent small cell lung cancer (SCLC) mouse models

在完全免疫活性的小细胞肺癌 (SCLC) 小鼠模型中研究抗磷脂酰肌醇蛋白聚糖 2 (GPC2) 嵌合抗原受体 (CAR) T 细胞运输和肿瘤微环境 (TME) 相互作用

• 批准号: 439733641
• 负责人: Dr. Hyatt Balke-Want
• 金额: --
• 依托单位: Klinik für Innere Medizin I
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/439733641?language=en
• 关键词: Investigating; anti; Glypican; GPC2; chimeric

SCLC is a highly lethal malignancy. It accounts for approximately 35,000 new cancer cases per year in the United States and 5-year overall survival rates are less than 7%. Still, first-line treatment of SCLC has not changed over the past decades and still relies on platinum-based chemotherapeutic regimens and radiation therapy1. Nevertheless, the extensive efforts that have been made to profoundly characterize SCLC on a genomic and transcriptomic level have lead to identification of relevant oncogenic lesions2. Among them are amplifications of the MYC family genes that can be found in 19% of SCLC3. MYC genes belong to the family of basic helix-loop-helix (bHLH) leucine zipper transcription factors, which are involved in cell cylce progression and proliferation4. So far, direct as well as indirect targeting of MYC familiy members in SCLC has been difficult5. However, in neuroblastoma (NB), where MYCN amplification is frequent, Dr. Crystal Mackall amongst others has identified anti-GPC2 directed antibody-drug conjugates and CAR T-cells to be effective in killing NB cells6,7. Given the recent advances in the field of CAR T-cell therapy including complete and durable responses even in highly aggressive leukemia and lymphoma8–10, I aim at applying anti-GPC2 CAR T-cells for treatment of SCLC.To ensure that GPC2 is a relevant target in SCLC as well, I will analyze GPC2 dependency in the context of MYC/MYCN/MYCL1 amplifications, overexpression and dependency (Aim 1). Notwithstanding, in light of the vastly immunosuppressive TME in SCLC that is capable of creating a functional barrier for CAR T-cells11,12, I will test anti-GPC2 CAR T-cells in genetically engineered mouse models (GEMMs) of SCLC in order to proof efficacy of anti-GPC2 CAR T-cells in fully immunocompetent and autochtonous models of SCLC. SCLC GEMMs will serve as a plattform to further characterize mediators of (un-) succesful anti-GPC2 CAR T-cell trafficking (Aim 2) and interactions of anti-GPC2 CAR T-cells with the immunosuppressive TME in SCLC (Aim 3). In order to succeed with Aim 2 we plan to stain CAR T-cells on tumor tissue of treated mice for identification of (in-)efficient tumor trafficking. For further characterization anti-GPC2 CAR T-cells and tumor endothelial cells will be introduced to CyTOF analysis to identify chemokine receptors and adehsion molecules expressed on their surfaces. Aim 3 will be approached applying single-cell RNA sequencing for identification of cellular components of the TME and their functional state. This multi-pronged approach will help us to derive a detailed understanding of the molecular features and cellular interactions that are necessary for sufficient anti-GPC2 CAR T-cell trafficking and for anti-GPC2 CAR T-cells to successfully conquere the immunosuppressive TME in SCLC. Our work will not only benefit treatment of SCLC, but will guide future CAR T-cell therapy for solid tumors in general.

SCLC是一种高度致命的恶性肿瘤。在美国，每年约有35，000例新发癌症病例，5年总生存率低于7%。尽管如此，SCLC的一线治疗在过去几十年中没有改变，仍然依赖于基于铂的化疗方案和放射治疗1。然而，在基因组和转录组水平上深入表征SCLC的广泛努力已经导致了相关致癌病变的鉴定2。其中包括MYC家族基因的扩增，可以在19%的SCLC 3中找到。MYC基因属于碱性螺旋-环-螺旋（bHLH）亮氨酸拉链转录因子家族，其参与细胞周期进展和增殖4。到目前为止，在小细胞肺癌中直接和间接针对MYC家族成员一直很困难5。然而，在MYCN扩增频繁的神经母细胞瘤（NB）中，Crystal Mackall博士等人已经鉴定出抗GPC 2定向的抗体-药物缀合物和CAR T细胞有效杀死NB细胞6，7。鉴于CAR T细胞治疗领域的最新进展，包括即使在高度侵袭性白血病和淋巴瘤中也能获得完全和持久的反应8 -10，我的目标是应用抗GPC 2 CAR T细胞治疗SCLC。为了确保GPC 2也是SCLC的相关靶标，我将在MYC/MYCN/MYCL 1扩增、过表达和依赖性的背景下分析GPC 2依赖性（目标1）。尽管如此，鉴于SCLC中的极大免疫抑制性TME能够为CAR T细胞产生功能屏障11，12，我将在SCLC的基因工程小鼠模型（GEMM）中测试抗GPC 2 CAR T细胞，以证明抗GPC 2 CAR T细胞在SCLC的完全免疫活性和自体模型中的功效。SCLC GEMM将作为进一步表征（未）成功的抗GPC 2 CAR T细胞运输（Aim 2）和抗GPC 2 CAR T细胞与SCLC中免疫抑制性TME相互作用（Aim 3）的介质的平台。为了成功实现目标2，我们计划对经治疗小鼠的肿瘤组织上的CAR T细胞进行染色，以鉴定（无效）肿瘤运输。为了进一步表征，将抗GPC 2 CAR T细胞和肿瘤内皮细胞引入CyTOF分析以鉴定在其表面上表达的趋化因子受体和粘附分子。目标3将应用单细胞RNA测序来鉴定TME的细胞组分及其功能状态。这种多管齐下的方法将帮助我们详细了解足够的抗GPC 2 CAR T细胞运输和抗GPC 2 CAR T细胞成功克服SCLC中免疫抑制性TME所必需的分子特征和细胞相互作用。我们的工作不仅有利于SCLC的治疗，而且将指导未来的CAR T细胞治疗实体瘤。