CAR T cell micropharmacies: Antigen-induced secretion of soluble VEGFR2 inhibitor for effective T cell invasion into solid cancers

嵌合抗原受体(CAR)工程T细胞有效靶向实体肿瘤的主要障碍是缺乏T细胞向肿瘤微环境(TME)的转运，以及TME免疫抑制成分的局部失活。该项目开发了一种创新的策略，通过整合分泌VEGFR2拮抗剂来放大基因工程T细胞的抗肿瘤活性。我们假设局部抑制VEGFR2将允许T细胞渗透，并促进其在TME中的功能，从而导致肿瘤根除。这一假说是基于VEGFR2在阻止T细胞渗入实体肿瘤和创造敌意的肿瘤微环境中的关键作用。T细胞将被改造成表达针对肿瘤相关抗原GD2的CAR，以及编码针对VEGFR2的抑制性抗原片段的基因。为了在肿瘤微环境中局部丰富VEGFR抑制物，将使用单载体方法通过CAR介导的T细胞激活来诱导产生。修饰后的T细胞能够溶解GD2+肿瘤细胞，并以抗原特异性、CAR诱导的方式产生和释放抑制性VEGFR2特异性抗体片段，这一能力将在体外得到证实。在小鼠模型中，我们将研究抗VEGFR2的释放在多大程度上促进T细胞向肿瘤血管系统的渗透，以及它如何影响TME的髓细胞成分。最后，我们将评估GD2CAR/抗VEGFR2基因修饰的T细胞在尤文肉瘤和神经母细胞瘤体内的抗肿瘤活性。从长远来看，该项目正在为该新策略的安全和有效的临床应用奠定基础。

Major barriers for effective targeting of solid tumors by chimeric antigen receptor (CAR) engineered T cells are the lack of T cell trafficking into the tumor microenvironment (TME) and their local inactivation by immunosuppressive components of the TME. This project develops an innovative strategy to amplify the anti-tumor activity of gene-engineered T cells by integrated secretion of VEGFR2 antagonist. We hypothesize that local inhibition of VEGFR2 will allow T cell infiltration and promote their function in the TME to result in tumor eradication. The hypothesis is based on the critical role of VEGFR2 in impeding T cell extravasation into solid tumors and creating a hostile tumor microenvironment. T cells will be engineered to express a CAR against the tumor-associated antigen GD2, along with the gene encoding for an inhibitory antigen fragment against VEGFR2. To locally enrich the VEGFR inhibitor in the tumor microenvironment, production will be inducible by CAR-mediated T cell activation, using a one-vector approach. The capacity of the modified T cells to lyse GD2+ tumor cells and produce and release inhibitory VEGFR2-specific antibody fragments in an antigen-specific, CAR-inducible manner will be demonstrated in vitro. In a mouse model, we will investigate to what extent release of anti-VEGFR2 promotes T cell infiltration into the tumor vasculature and how it affects myeloid cell components of the TME. Finally, we will assess the therapeutic antitumor activity of GD2 CAR/anti-VEGFR2 gene-modified T cells in in vivo models of Ewing sarcoma and neuroblastoma. Perspectively, this project is establishing the prerequisites for safe and effective clinical application of the novel strategy.