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

SCLC是一种高度致命的恶性肿瘤。在美国，每年大约有35000例新的癌症病例，5年总生存率不到7%。然而，在过去的几十年里，SCLC的一线治疗并没有改变，仍然依赖于以铂为基础的化疗方案和放疗。尽管如此，在基因组和转录组学水平上深入表征SCLC的广泛努力已经导致了相关致癌病变的鉴定2。其中有19%的scc3患者存在MYC家族基因的扩增。MYC基因属于碱性螺旋-环-螺旋（bHLH）亮氨酸拉链转录因子家族，参与细胞周期的进展和增殖4。到目前为止，在SCLC中直接或间接靶向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博士等人发现抗gpc2导向的抗体-药物偶联物和CAR - t细胞可有效杀死NB细胞。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有完全和持久的反应8 - 10，我的目标是应用抗gpc2 CAR - t细胞治疗SCLC。为了确保GPC2在SCLC中也是一个相关的靶点，我将分析GPC2在MYC/MYCN/MYCL1扩增、过表达和依赖的背景下的依赖性（目的1）。尽管如此，鉴于SCLC中的TME具有巨大的免疫抑制作用，能够为CAR - t细胞创造功能屏障11,12，我将在SCLC的基因工程小鼠模型（GEMMs）中测试抗gpc2 CAR - t细胞，以证明抗gpc2 CAR - t细胞在完全免疫能力和自体SCLC模型中的有效性。SCLC gem将作为一个平台，进一步表征（非）成功的抗gpc2 CAR - t细胞运输（Aim 2）和抗gpc2 CAR - t细胞与SCLC免疫抑制TME的相互作用（Aim 3）。为了在Aim 2中取得成功，我们计划在治疗小鼠的肿瘤组织上染色CAR - t细胞，以鉴定（In -）有效的肿瘤运输。为了进一步表征抗gpc2 CAR - t细胞和肿瘤内皮细胞，将引入CyTOF分析，以鉴定趋化因子受体及其表面表达的粘附分子。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帮助我们详细了解足够的抗gpc2 CAR - t细胞运输和抗gpc2 CAR - t细胞成功征服SCLC免疫抑制性TME所必需的分子特征和细胞相互作用。我们的工作不仅将有利于SCLC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.