同时靶向 Tim3 和 A2a 受体可调节 MSLN-CAR T 细胞在人宫颈肿瘤异种移植模型中的抗肿瘤功能。

INTRODUCTION: 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) and METHODS: 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 through coculture with cervical cancer cells (HeLa cell line). To evaluate antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. RESULTS: 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. CONCLUSION: 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.

简介：嵌合抗原受体 (CAR) T 细胞疗法已经改变了血液恶性肿瘤的治疗方式。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，这在临床环境中损害了 CAR T 细胞的抗肿瘤功能。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（包括 A2aR（腺苷 A2 受体）和 Tim3（T 细胞免疫球蛋白和粘蛋白结构域蛋白 3））的潜力，以增强 CAR T 细胞的功能。在这项研究中，我们评估了基因靶向 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。

方法：使用标准细胞和分子技术生产第二代抗间皮素 CAR T 细胞。使用 shRNA 介导的基因沉默生成 A2aR 敲低和/或 Tim3 敲低的抗间皮素-CAR T 细胞。通过与宫颈癌细胞（HeLa 细胞系）共培养来评估 CAR T 细胞的抗肿瘤功能，测量细胞因子产生、增殖和细胞毒性。为了评估制造的 CAR T 细胞的抗肿瘤疗效，在人宫颈癌异种移植模型中监测肿瘤生长和小鼠存活情况。

结果：实验表明，单独敲低 A2aR 或与 Tim3 联合敲低以抗原特异性方式显著改善了 CAR T 细胞在肿瘤细胞存在时的增殖、细胞因子产生和细胞毒性。此外，在人源化异种移植模型中，双敲低 CAR T 细胞和对照 CAR T 细胞均能有效控制肿瘤生长。然而，单独敲低 CAR T 细胞与小鼠存活减少相关。

结论：这些发现强调了同时基因靶向 Tim3 和 A2a 受体以增强 CAR T 细胞疗法在实体瘤中的疗效的潜力。然而，鉴于我们观察到单独敲低 MSLN-CAR T 细胞治疗的小鼠存活减少，应谨慎行事，强调需要仔细考虑疗效。