靶向CD5嵌合抗原受体工程化自然杀伤细胞治疗T细胞恶性肿瘤。
Targeting CD5 chimeric antigen receptor-engineered natural killer cells against T-cell malignancies.
作者信息
Zu Yingling, Ren Quan, Zhang Jishuai, Su Hongchang, Lu Qiumei, Song Yongping, Zhou Jian
机构信息
Department of Hematology, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou, Henan, 450008, China.
Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, China.
出版信息
Exp Hematol Oncol. 2024 Oct 26;13(1):104. doi: 10.1186/s40164-024-00577-5.
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.
背景
嵌合抗原受体工程化T细胞（CAR-T）在B细胞恶性肿瘤中已显示出有前景的临床疗效，并且该方法已扩展至T细胞恶性肿瘤。然而，由于存在移植物抗宿主病的风险，在CAR治疗中使用同种异体T细胞带来了挑战。最近，自然杀伤（NK）细胞表现出“现成可用”的特性。基于纳米抗体的CAR结构因其纳米抗体的优势，包括尺寸小、稳定性佳、亲和力高和易于制造，而在治疗潜力方面备受关注。CD5是恶性T细胞的常见表面标志物，其细胞外区域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源自纳米抗体的CD5膜近端结构域的“现成可用”CAR-NK细胞，用于治疗T细胞恶性肿瘤。
方法
通过噬菌体展示技术筛选抗CD5-D3纳米抗体，随后构建可异位产生IL-15的第四代CAR质粒，以生成源自外周血的CD5 CAR-NK细胞。并生成基于纳米抗体的第二代CD5 CAR-T细胞，称为5D.b CAR-T和12C.b CAR-T。此外，生成不含IL-15的CAR-NK细胞（IL-15 CAR-NK），以评估其对CAR-NK细胞细胞毒性的影响。在体外对CD5血液恶性细胞系和正常T细胞发挥细胞毒活性，并使用移植了Jurkat-Luc细胞的NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt小鼠模型在体内评估CD5 CAR-NK细胞的抗肿瘤疗效。
结果
两种纳米抗体（5D和12C）竞争结合CD5-D3的表位。12C CAR-NK细胞在抗肿瘤潜力方面优于5D CAR-NK细胞，并且12C.b CAR-T细胞在体外表现出比5D CAR-T细胞更强的细胞毒活性。因此，12C被视为最佳纳米抗体。12C CAR-NK细胞和IL-15 CAR-NK细胞对CD5恶性细胞系表现出强大的细胞毒性，并在异种移植小鼠模型中控制了疾病进展。在体外和体内，12C CAR-NK细胞均显示出比IL-15 CAR-NK细胞更强的抗肿瘤活性。
结论
综上所述，第四代纳米抗体衍生的抗CD5 CAR-NK细胞可能是一种有前景的T细胞恶性肿瘤治疗方法。
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