肿瘤细胞治疗研究
英文原题：Targeting CD5 chimeric antigen receptor-engineered natural killer cells against T-cell malignancies.
第四代纳米抗体来源的抗 CD5 CAR-NK 细胞可能成为治疗 T 细胞恶性肿瘤的有前景的疗法。
背景：嵌合抗原受体工程化T细胞（CAR-T）在B细胞恶性肿瘤中显示出令人鼓舞的临床疗效，该策略也已拓展至T细胞恶性肿瘤。然而，CAR疗法使用异体T细胞面临移植物抗宿主病风险。近期，天然杀伤（NK）细胞展现出“现货型”治疗的潜力。基于纳米抗体的CAR结构因纳米抗体具有体积小、稳定性良好、亲和力高且易于生产等优势，其治疗潜力受到广泛关注。CD5是恶性T细胞常见的表面标志物，其胞外区含有三个富含半胱氨酸的清道夫受体结构域（D1-D3）。本研究旨在构建靶向CD5膜近端结构域、源自抗体纳米抗体的“现货型”CAR-NK细胞，用于治疗T细胞恶性肿瘤。 方法：通过噬菌体展示筛选抗CD5-D3纳米抗体，随后构建可异位表达IL-15的第四代CAR质粒，以制备来源于外周血的CD5 CAR-NK细胞。同时，构建基于纳米抗体的第二代CD5 CAR-T细胞，分别称为5D.b CAR-T和12C.b CAR-T。此外，还制备不含IL-15的CAR-NK细胞（IL-15 CAR-NK），以评估IL-15对CAR-NK细胞细胞毒作用的影响。我们在体外评估其对CD5阳性血液系统恶性肿瘤细胞系和正常T细胞的细胞毒活性，并使用移植Jurkat-Luc细胞的NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt小鼠模型评估CD5 CAR-NK细胞的体内抗肿瘤疗效。 结果：两种纳米抗体（5D和12C）均可竞争性结合CD5-D3表位。12C CAR-NK细胞的抗肿瘤潜力优于5D CAR-NK细胞；在离体实验中，12C.b CAR-T细胞的细胞毒活性也优于5D CAR-T细胞。因此，12C被确定为最佳纳米抗体。12C CAR-NK和IL-15 CAR-NK细胞均对CD5阳性恶性细胞系表现出强劲的细胞毒作用，并能控制异种移植小鼠模型中的疾病进展。在体外和体内，12C CAR-NK细胞的抗肿瘤活性均高于IL-15 CAR-NK细胞。 结论：综上，基于纳米抗体的第四代抗CD5 CAR-NK细胞可能成为治疗T细胞恶性肿瘤的一种有前景的疗法。
BACKGROUND: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated promising clinical efficacy in B-cell malignancies, and the approach has been extended to T-cell malignancies. However, the use of allogeneic T cells in CAR therapy poses a challenge due to the risk of graft-versus-host disease. Recently, natural killer (NK) cells have exhibited "off the shelf" availability. The nanobody-based CAR structures have attracted much attention for their therapeutic potential owing to the advantages of nanobody, including small size, optimal stability, high affinity and manufacturing feasibility. CD5, a common surface marker of malignant T cells, has three scavenger receptor cysteine-rich domains (D1-D3) in the extracellular region. The present study aims to construct "off the shelf" CAR-NK cells targeting the membrane-proximal domain of CD5 derived from nanobody against T-cell malignancies. METHODS: Anti-CD5-D3 nanobody was screened by phage display technology, followed by constructing fourth-generation CAR plasmids ectopically producing IL-15 to generate CD5 CAR-NK cells derived from peripheral blood. And the second-generation CD5 CAR-T cells based on nanobody were generated, referred to as 5D.b CAR-T and 12 C.b CAR-T. Furthermore, CAR-NK cells without IL-15 (IL-15 CAR-NK) were generated to assess the impact on cytotoxicity of CAR-NK cells. Cytotoxic activity against CD5 + hematologic malignant cell lines and normal T cells was exerted in vitro and NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mouse model transplanted with Jurkat-Luc cells was used to evaluate the antitumor efficacy of CD5 CAR-NK cells in vivo. RESULTS: Two nanobodies (5D and 12 C) competed for binding to the epitope of CD5-D3. 12 C CAR-NK cells were superior to 5D CAR-NK cells in antitumor potential and 12 C.b CAR-T cells exhibited superior cytotoxic activity than 5D CAR-T cells ex vivo. So, 12 C was regarded as the optimal nanobody. 12 C CAR-NK cells and IL-15 CAR-NK cells exhibited robust cytotoxicity against CD5 + malignant cell lines and controlled disease progression in xenograft mouse model. 12 C CAR-NK cells demonstrated greater antitumor activity compared to that of IL-15 CAR-NK cells in vitro and in vivo. CONCLUSIONS: Taken together, the fourth-generation nanobody-derived anti-CD5 CAR-NK cells may be a promising therapeutic against T-cell malignancies.
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