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

小细胞肺癌是一种高度致命的恶性肿瘤。在美国，每年大约有35,000个新的癌症病例，5年的总存活率不到7%。尽管如此，小细胞肺癌的一线治疗在过去几十年中没有改变，仍然依赖于以铂为基础的化疗方案和放射治疗1。然而，为在基因组和转录水平上深刻描述小细胞肺癌所做的广泛努力导致了相关致癌损伤的鉴定2。其中有MYC家族基因的扩增，可在19%的SCLC 3中发现。MYC基因属于碱性螺旋-环-螺旋(BHLH)亮氨酸拉链转录因子家族，参与细胞的生长和增殖。到目前为止，在小细胞肺癌中直接和间接靶向MYC家族成员都是困难的。然而，在MYCN扩增频繁的神经母细胞瘤(NB)中，Crystal Mackall博士和其他人已经发现抗GPC2导向的抗体药物结合物和CAR T细胞可以有效地杀死NB细胞6，7。鉴于CAR T细胞治疗领域的最新进展，包括即使在高侵袭性白血病和淋巴瘤8-10中也能产生完全和持久的反应，我打算将抗GPC2 CAR T细胞应用于SCLC的治疗。为了确保GPC2也是SCLC的相关靶点，我将在MYC/MYCN/MYCL1扩增、过度表达和依赖的背景下分析GPC2依赖。尽管如此，鉴于SCLC中的TME具有极大的免疫抑制作用，能够为CAR T细胞11、12创造功能障碍，我将在SCLC的基因工程小鼠模型(GEMM)中测试抗GPC2 CAR T细胞，以证明抗GPC2 CAR T细胞在完全免疫活性和自体SCLC模型中的有效性。SCLC GEMMS将作为一个平台，进一步表征(不)成功的抗GPC2 CAR T细胞转运的介体(AIM 2)以及抗GPC2 CAR T细胞与SCLC中免疫抑制的TME的相互作用(AIM 3)。为了成功实现目标2，我们计划在治疗小鼠的肿瘤组织上染色CAR T细胞，以鉴定(无效)肿瘤的转运。为了进一步鉴定，我们将把抗GPC2的CAR T细胞和肿瘤内皮细胞引入细胞周期分析，以鉴定其表面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的细胞成分及其功能状态。这种多管齐下的方法将有助于我们详细了解足够的抗GPC2 CAR T细胞运输和抗GPC2 CAR T细胞成功克服小细胞肺癌免疫抑制TME所必需的分子特征和细胞相互作用。我们的工作不仅将有益于小细胞肺癌的治疗，而且将指导未来实体瘤的CAR T细胞治疗。

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.