FGFR4-CAR-based Immunotherapy of Fusion-Positive Rhabdomyosarcoma

项目摘要/摘要 横纹肌肉瘤(RMS)是一种毁灭性的儿科恶性肿瘤，几十年来一直没有好转。 适用于高危或复发的患者。这种疾病的一个子集，ARMS，通常更严重， 由PAX3/7-FOXO1融合基因产物驱动，具有极少的其他突变，并表达 表面有FGFR4蛋白。在之前的工作中，我们使用了广泛的基因组分析来定义 FGFR4作为CAR-T目标，并使用scFv表达文库创建了一组针对FGFR4的CARS。 其中两种结合剂在体外分离了功能，其中一种在体内静脉注射时部分有效。NSG中的模型 小鼠(注射我们设计的也表达CD19的Rh30细胞系)。死于静脉注射的小鼠。肿瘤 也有肝转移，间质丰富，诱导PD-L1表达，这在肿瘤中是不存在的。 小鼠既没有接受CAR-T，也没有接受激活的非转导T细胞。重要的是，当 肌肉注射Rh30_19，未见肿瘤控制。根据组织学分析，肝脏蛋氨酸和I.M. 肿瘤具有典型的“免疫排斥”形态，即存在CD4和CD8淋巴细胞，但 主要局限于肿瘤周边。这导致我们在此应用程序中提出了新的方法来 改进手臂的CAR-T疗法。当我们绘制当前基于scFv的汽车的结合位置时，它是 在靶细胞膜的很远的地方。我们早先对CD22的研究表明，一辆更活跃的汽车可以 通过靶向膜近端结构域来创建。在这里，我们将使用工程化结构域抗体(Dab)。 而不是单链抗体来靶向FGFR4上的新表位。迪米特罗夫实验室独有的DAB是基于稳定的， 可溶的仅VH结构域，并以工程噬菌体库格式展示，具有令人难以置信的大 多样性。DAB在大小上比scFv小，并能够唯一地识别目标上的新结合位点 蛋白质。我们概述了一种针对FGFR4更接近膜的结构域的方法。其次，我们 装甲汽车将颠覆PD-1结合、转化生长因子-β和Fas诱导的负面信号。第三，我们 会通过胶原酶的使用破坏肿瘤间质的物理结构和生物形成 和全反式维甲酸(ATRA)。胶原酶打破胶原蛋白对免疫的物理屏障 细胞，并释放隔离在基质基质中的趋化因子。全反式维甲酸直接影响髓系细胞 抑制细胞在肿瘤微环境中的活性，并使MDSC较少受到免疫抑制。穿过 靶向膜近端的FGFR4结构域，装甲CAR，并破坏肿瘤组织 微环境我们建议创造新一代FGFR4专用汽车，将影响这一可怕的 儿科癌症。

PROJECT SUMMARY/ABSTRACT Rahbdomyosarcoma (RMS) is a devastating pediatric malignancy that has not shown improvement in decades for patients with high-risk or recurrent disease. One subset of this disease, ARMS, is usually more severe, is driven by a PAX3/7-FOXO1 fusion gene product, has remarkably few other mutations, and expresses the protein FGFR4 on its surface. In previous work we used extensive genomic analysis to define the relevance of FGFR4 as a CAR-T target and created a set of CARs that target FGFR4 using an scFv expression library. Two of the binders isolated function in vitro, and one was partially effective in vivo using an i.v. model in NSG mice (injection of the Rh30 cell line we engineered to also express CD19). Mice that succumbed to i.v. tumor also had liver metastases with a rich stroma and induced expression of PD-L1, which was not seen in tumor- bearing mice that did not receive either CAR-T or activated non-transduced T cells. Importantly, when Rh30_19 was injected i.m., no tumor control was seen. Upon histological analysis, both liver mets and i.m. tumor had a classic “immune excluded” morphology, in that CD4 and CD8 lymphocytes were present, but primarily were restricted to the tumor periphery. This led us to propose new approaches in this application to improve CAR-T therapy for ARMS. When we mapped the binding site of our current scFv-based CAR, it was quite distal to the target cell membrane. Our earlier work with CD22 demonstrated that a more active CAR can be created by targeting membrane proximal domains. Here we will use engineered domain antibodies (dAb) instead of scFv to target new epitopes on FGFR4. Unique to the Dimitrov lab, dAb are based on a stable, soluble, VH-only domain, and are displayed in an engineered phage library format with incredible large diversity. dAb are smaller in size than scFv and are able to uniquely identify new binding sites on target proteins. We outline an approach that will target a more membrane proximal domain of FGFR4. Secondly, we will armor CARs to subvert the negative signals induced by PD-1 binding, by TGF-beta, and by Fas. Third, we will disrupt the physical structure and the biological formation of tumor stroma through the use of collagenase and all-trans retinoic acid (ATRA). Collagenase breaks down the physical barrier collagen presents to immune cells, and releases chemokines sequestered in the stromal matrix. ATRA directly impacts myeloid-derived suppressor cells activity in the tumor micorenvironment and renders MDSC less immunosuppressive. Through the targeting of a membrane proximal FGFR4 domain, armoring of the CAR, and disruption of the tumor tissue microenvironment we propose to create a new generation of FGFR4-specific CARs that will impact this dire pediatric cancer.