Abstract：
OBJECTIVE: Chimeric antigen receptor T (CAR-T) cells targeting single antigens show limited activity against solid tumors due to poor T cell persistence, low efficiency infiltration, and exhaustion together with heterogeneous tumor-associated antigen (TAA) expression. This is also true in high-risk neuroblastoma (HRNB), a lethal pediatric extracranial malignancy. To overcome these obstacles, a combinational strategy using GD2-specific and GPC2-specific CAR-T cells was developed to improve immunotherapeutic efficacy.
METHODS: We individually developed GD2-specific and GPC2-specific CARs containing a selective domain (sCAR) which was a peptide of 10 amino acids derived from human nuclear autoantigen La/SS-B. These constructs allowed us to generate two different HRNB antigen-specific CAR-T cells with enhanced biological activity through stimulating sCAR-engrafted T cells via a selective domain-specific monoclonal antibody (SmAb). Binding affinity and stimulation of GD2- and GPC2-specific sCARs by SmAb were measured, and transient and persistent anti-tumor cytotoxicity of GD2sCAR-T and GPC2sCAR-T cells were quantified in neuroblastoma cell lines expressing different TAA levels. The anti-tumor pharmaceutical effects and cellular mechanisms mediated by single or combinational sCAR-T cells were evaluated in vitro and in vivo.
RESULTS: GD2- and GPC2-specific sCARs had antigen-specific binding affinity similar to their parental counterparts and were recognized by SmAb. SmAb-mediated stimulation selectively activated sCAR-T proliferation and increased central memory T cells in the final products. SmAb-stimulated sCAR-T cells had enhanced transient cytolytic activity, and combination therapy extended long-term anti-tumor activity in vitro through TNF-α and IL-15 release. Stimulated sCAR-T cells overcame heterogeneous antigen expression in HRNB, and the multi-TAA-targeting strategy was especially efficacious in vivo, inducing apoptosis through the caspase-3/PARP pathway and inhibiting the release of several tumor-promoting cytokines.
CONCLUSIONS: These data suggest that combined targeting of multiple TAAs is a promising strategy to overcome heterogenous antigen expression in solid tumors and extend CAR-T cell persistence for HRNB immunotherapy.
METHODS: We individually developed GD2-specific and GPC2-specific CARs containing a selective domain (sCAR) which was a peptide of 10 amino acids derived from human nuclear autoantigen La/SS-B. These constructs allowed us to generate two different HRNB antigen-specific CAR-T cells with enhanced biological activity through stimulating sCAR-engrafted T cells via a selective domain-specific monoclonal antibody (SmAb). Binding affinity and stimulation of GD2- and GPC2-specific sCARs by SmAb were measured, and transient and persistent anti-tumor cytotoxicity of GD2sCAR-T and GPC2sCAR-T cells were quantified in neuroblastoma cell lines expressing different TAA levels. The anti-tumor pharmaceutical effects and cellular mechanisms mediated by single or combinational sCAR-T cells were evaluated in vitro and in vivo.
RESULTS: GD2- and GPC2-specific sCARs had antigen-specific binding affinity similar to their parental counterparts and were recognized by SmAb. SmAb-mediated stimulation selectively activated sCAR-T proliferation and increased central memory T cells in the final products. SmAb-stimulated sCAR-T cells had enhanced transient cytolytic activity, and combination therapy extended long-term anti-tumor activity in vitro through TNF-α and IL-15 release. Stimulated sCAR-T cells overcame heterogeneous antigen expression in HRNB, and the multi-TAA-targeting strategy was especially efficacious in vivo, inducing apoptosis through the caspase-3/PARP pathway and inhibiting the release of several tumor-promoting cytokines.
CONCLUSIONS: These data suggest that combined targeting of multiple TAAs is a promising strategy to overcome heterogenous antigen expression in solid tumors and extend CAR-T cell persistence for HRNB immunotherapy.
摘要：
目的：由于T细胞持久性差，靶向单一抗原的嵌合抗原受体T（CAR-T）细胞对实体瘤的活性有限，低效率渗透，与异质性肿瘤相关抗原（TAA）表达一起耗尽。在高风险神经母细胞瘤（HRNB）中也是如此，一种致命的小儿颅外恶性肿瘤.为了克服这些障碍,开发了一种使用GD2特异性和GPC2特异性CAR-T细胞的组合策略,以提高免疫治疗效果.
方法：我们单独开发了GD2特异性和GPC2特异性CAR，其中包含选择性结构域（sCAR），该选择性结构域是源自人核自身抗原La/SS-B的10个氨基酸的肽。这些构建体使我们能够通过经由选择性结构域特异性单克隆抗体(SmAb)刺激sCAR移植的T细胞来产生具有增强的生物活性的两种不同的HRNB抗原特异性CAR-T细胞。测量了SmAb对GD2-和GPC2特异性sCAR的结合亲和力和刺激，在表达不同TAA水平的神经母细胞瘤细胞系中定量GD2sCAR-T和GPC2sCAR-T细胞的瞬时和持续抗肿瘤细胞毒性。在体外和体内评估了由单个或组合sCAR-T细胞介导的抗肿瘤药物作用和细胞机制。
结果：GD2-和GPC2特异性sCAR具有与其亲本对应物相似的抗原特异性结合亲和力，并被SmAb识别。SmAb介导的刺激选择性激活最终产物中的sCAR-T增殖并增加中枢记忆T细胞。SmAb刺激的sCAR-T细胞具有增强的瞬时细胞溶解活性，联合治疗通过TNF-α和IL-15的释放延长了体外长期抗肿瘤活性。刺激的sCAR-T细胞克服了HRNB中的异质性抗原表达，多TAA靶向策略在体内特别有效，通过caspase-3/PARP途径诱导细胞凋亡并抑制几种促肿瘤细胞因子的释放。
结论：这些数据表明，多个TAA的联合靶向是克服实体肿瘤中异质性抗原表达并延长CAR-T细胞持久性的有希望的策略用于HRNB免疫治疗。
方法：我们单独开发了GD2特异性和GPC2特异性CAR，其中包含选择性结构域（sCAR），该选择性结构域是源自人核自身抗原La/SS-B的10个氨基酸的肽。这些构建体使我们能够通过经由选择性结构域特异性单克隆抗体(SmAb)刺激sCAR移植的T细胞来产生具有增强的生物活性的两种不同的HRNB抗原特异性CAR-T细胞。测量了SmAb对GD2-和GPC2特异性sCAR的结合亲和力和刺激，在表达不同TAA水平的神经母细胞瘤细胞系中定量GD2sCAR-T和GPC2sCAR-T细胞的瞬时和持续抗肿瘤细胞毒性。在体外和体内评估了由单个或组合sCAR-T细胞介导的抗肿瘤药物作用和细胞机制。
结果：GD2-和GPC2特异性sCAR具有与其亲本对应物相似的抗原特异性结合亲和力，并被SmAb识别。SmAb介导的刺激选择性激活最终产物中的sCAR-T增殖并增加中枢记忆T细胞。SmAb刺激的sCAR-T细胞具有增强的瞬时细胞溶解活性，联合治疗通过TNF-α和IL-15的释放延长了体外长期抗肿瘤活性。刺激的sCAR-T细胞克服了HRNB中的异质性抗原表达，多TAA靶向策略在体内特别有效，通过caspase-3/PARP途径诱导细胞凋亡并抑制几种促肿瘤细胞因子的释放。
结论：这些数据表明，多个TAA的联合靶向是克服实体肿瘤中异质性抗原表达并延长CAR-T细胞持久性的有希望的策略用于HRNB免疫治疗。
