PDF(Pubmed)
Abstract：
UNASSIGNED: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo.
UNASSIGNED: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model.
UNASSIGNED: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice.
UNASSIGNED: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.
UNASSIGNED: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model.
UNASSIGNED: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice.
UNASSIGNED: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.
摘要：
■嵌合抗原受体（CAR）T细胞疗法已改变了血液恶性肿瘤的治疗方法。然而,其在实体瘤中的疗效受到免疫抑制性肿瘤微环境的限制,该环境损害了临床环境中的CART细胞抗肿瘤功能.为了克服这一挑战，研究人员研究了抑制特定免疫检查点受体的潜力，包括A2aR（腺苷A2受体）和Tim3（T细胞免疫球蛋白和含粘蛋白结构域的蛋白3），以增强CAR-T细胞功能。在这项研究中,我们在体外和体内评估了基因靶向Tim3和A2a受体对人间皮素特异性CAR-T细胞(MSLN-CAR)抗肿瘤功能的影响.
■使用标准细胞和分子技术产生第二代抗间皮素CART细胞。使用shRNA介导的基因沉默产生A2aR-敲低和/或Tim3-敲低抗间皮素-CART细胞。通过测量细胞因子的产生来评估CART细胞的抗肿瘤功能。扩散,通过与宫颈癌细胞（HeLa细胞系）共培养在体外具有细胞毒性。为了评估制造的CART细胞的体内抗肿瘤功效，在人宫颈癌异种移植模型中监测肿瘤生长和小鼠存活。
■体外实验表明，单独敲除A2aR或与Tim3联合使用可显著提高CAR-T细胞增殖，细胞因子产生，和以抗原特异性方式存在肿瘤细胞的细胞毒性。此外，在人性化的异种移植模型中，双敲低CART细胞和对照CART细胞均能有效控制肿瘤生长。然而,单个敲低CART细胞与小鼠存活率降低有关。
■这些发现强调了伴随基因靶向Tim3和A2a受体以增强CAR-T细胞疗法在实体瘤中的功效的潜力。然而，根据我们观察到的单敲除MSLN-CAR-T细胞治疗的小鼠存活率降低，应谨慎行事。强调需要仔细考虑功效。
■使用标准细胞和分子技术产生第二代抗间皮素CART细胞。使用shRNA介导的基因沉默产生A2aR-敲低和/或Tim3-敲低抗间皮素-CART细胞。通过测量细胞因子的产生来评估CART细胞的抗肿瘤功能。扩散,通过与宫颈癌细胞（HeLa细胞系）共培养在体外具有细胞毒性。为了评估制造的CART细胞的体内抗肿瘤功效，在人宫颈癌异种移植模型中监测肿瘤生长和小鼠存活。
■体外实验表明，单独敲除A2aR或与Tim3联合使用可显著提高CAR-T细胞增殖，细胞因子产生，和以抗原特异性方式存在肿瘤细胞的细胞毒性。此外，在人性化的异种移植模型中，双敲低CART细胞和对照CART细胞均能有效控制肿瘤生长。然而,单个敲低CART细胞与小鼠存活率降低有关。
■这些发现强调了伴随基因靶向Tim3和A2a受体以增强CAR-T细胞疗法在实体瘤中的功效的潜力。然而，根据我们观察到的单敲除MSLN-CAR-T细胞治疗的小鼠存活率降低，应谨慎行事。强调需要仔细考虑功效。
