肿瘤细胞治疗研究
英文原题：Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice.
Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice.
我们的新型同基因CAR Tg小鼠模型可作为解决临床相关问题并开发未来免疫治疗策略的有用工具。
嵌合抗原受体（CAR）免疫治疗的严格临床前研究将需要大量一致且高质量的CAR转导T（CART）细胞，这些细胞可用于同基因小鼠胶质母细胞瘤（GBM）模型。为此，我们开发了一种新型转基因（Tg）小鼠品系，其携带完全鼠源化的靶向表皮生长因子受体变异体III（EGFRvIII）的CAR。
我们首先构建了EGFRvIII-CAR的小鼠化版本，并在表达EGFRvIII的同源SB28 GBM的C57BL/6J小鼠中使用逆转录病毒载体（RV）验证了其功能。接下来，我们创建了C57BL/6J背景的Tg小鼠，其在Rosa26位点的Lox-Stop-Lox盒下游携带抗EGFRvIII-CAR。我们将这些小鼠与CD4-Cre Tg小鼠交配，以使CAR在T细胞上表达，并在体外和体内评估了CART细胞的功能。为了抑制SB28 GBM内的免疫抑制性髓系细胞，我们还评估了CART与抗EP4化合物（ONO-AE3-208）的联合方法。
RV-和Tg-CART细胞均显示出对SB28-EGFRvIII细胞的特异性细胞毒活性。在淋巴细胞清除方案预处理后，单次静脉输注EGFRvIII-CART细胞延长了荷胶质瘤小鼠的生存期，复发肿瘤表现出显著的EGFRvIII丢失。加入ONO-AE3-208后，一部分接受CART治疗的小鼠实现了长期生存，这些存活小鼠在皮下再次接种EGFRvIII+和亲本EGFRvIII- SB28后均表现出肿瘤生长延迟。
BACKGROUND: Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). METHODS: We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). RESULTS: Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. CONCLUSION: Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.
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