Targeting Osteosarcoma with Canine B7-H3 CAR T Cells and Impact of CXCR2 Co-Expression on Functional Activity. Targeting Osteosarcoma with Canine B7-H3 CAR T Cells and Impact of CXCR2 Co-Expression on Functional Activity

Abstract. The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. Therefore, the goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS and glioma in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed high levels of B7-H3, whereas levels were undetectable on normal dog tissues. In vitro, both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little antitumor activity was generated by B7-H3 CAR T cells, whereas B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept. Overall design: IN the current study we expanded the scope of the original canine B7-H3 CAR T cell studies to evaluate expression of B7-H3 using a larger panel of canine OS tumor cell lines and biopsy tissues. In addition, these studies conducted in vitro and in vivo characterization of a dual B7-H3 CAR construct co-expressing CXCR2.

摘要
利用大型动物自发性实体瘤模型（例如罹患骨肉瘤（osteosarcoma, OS）的犬只），有助于开发包括嵌合抗原受体（chimeric antigen receptor, CAR）T细胞在内的新型癌症免疫治疗策略。因此，本研究的核心目标为制备靶向B7-H3（CD276）共刺激分子的犬源CAR T细胞；该分子在人类与犬类的多种实体瘤（包括OS与胶质瘤）中均呈高表达，同时本研究将评估此类CAR T细胞识别犬OS细胞系或异种移植模型中犬肿瘤所表达的B7-H3的能力。本研究的另一项目标是探究：相较于仅表达B7-H3的CAR T细胞，共表达趋化因子受体的新型双特异性CAR是否可增强犬源CAR T细胞的抗肿瘤活性。
据此，本研究通过以下实验展开相关探究：检测犬OS肿瘤组织的B7-H3表达水平、体外评估犬源B7-H3 CAR T细胞的靶点结合能力，以及在犬异种移植模型中比较B7-H3 CAR T细胞与B7-H3-CXCR2双特异性CAR T细胞的相对疗效。
研究结果显示，多数犬OS肿瘤组织的B7-H3呈高表达，而正常犬组织中无法检测到该分子的表达。体外实验中，两类B7-H3 CAR T细胞均表现出活化特性与OS特异性杀伤能力，但B7-H3-CXCR2双特异性CAR T细胞的细胞因子分泌水平显著更高。在犬OS异种移植模型中，单纯B7-H3 CAR T细胞几乎未表现出抗肿瘤活性，而B7-H3-CXCR2双特异性CAR T细胞可显著抑制肿瘤生长，并使多数受试小鼠实现完全肿瘤清除。综上，共表达趋化因子受体可显著增强犬源B7-H3 CAR T细胞的抗肿瘤活性；因此，针对原发性或转移性OS患犬开展该新型双特异性CAR构建体的相关评估具有重要意义，此类研究可对趋化因子受体相关策略进行关键且贴合临床实际的验证。
整体实验设计：本研究拓展了前期犬源B7-H3 CAR T细胞相关研究的范围，通过纳入更多犬OS肿瘤细胞系与活检组织来评估B7-H3的表达水平；此外，本研究还通过体外与体内实验，对共表达CXCR2的B7-H3双特异性CAR构建体进行了功能表征。