Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.
Center for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
J Immunother Cancer. 2024 Apr 5;12(4):e008174. doi: 10.1136/jitc-2023-008174.
Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft.
We produced CB-CD8 T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCRα () and TCRβ () chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts.
The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR WT1-TCR CD8 CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells.
In summary, we show the feasibility of developing a potent CB-derived CD8 T cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.
造血细胞移植（HCT）是治疗高危、难治或复发急性髓系白血病（AML）患儿的有效方法。然而，很大一部分移植患者最终因复发而死亡。为了提高总生存率，我们提出了一种基于脐带血（CB）-HCT 的联合策略，并应用来自同一 CB 移植物的 AML 特异性 T 细胞受体（TCR）工程化 T 细胞治疗。
我们产生了表达针对 Wilms 肿瘤 1（WT1）的重组 TCR（rTCR）的 CB-CD8 T 细胞，同时缺乏内源性 TCR（eTCR）表达，以避免错配和竞争。CRISPR-Cas9 多重靶向内源性 TCRα（）和 TCRβ（）链的恒定区。接下来，我们使用优化的慢病毒转导方法引入重组 WT1-TCR。在细胞系和原发性儿科 AML 白血病细胞的共培养实验中，评估产物的细胞毒性和迁移能力。
基因编辑和转导过程效率高，高达 95%的细胞缺乏 eTCR，超过 70%的 T 细胞表达 rWT1-TCR。与仍表达其 eTCR（eTCR WT1-TCR）的 WT1-TCR 工程化 T 细胞相比，缺乏 eTCR 表达的 WT1-TCR 工程化 T 细胞（eTCR WT1-TCR）在抗原识别时表现出更高的 rTCR 细胞表面表达和细胞毒性细胞因子（如颗粒酶 A 和 B、穿孔素、干扰素-γ（IFNγ）和肿瘤坏死因子-α（TNFα））的产生。CRISPR-Cas9 编辑不影响免疫表型特征或 T 细胞激活，也不会诱导抑制分子的表达增加。eTCR WT1-TCR CD8 CB-T 细胞在与肿瘤细胞系和原发性 AML 白血病细胞的共培养中表现出有效的迁移和杀伤能力，但对健康细胞没有毒性。
总之，我们展示了开发针对 WT1 的有效 CB 衍生 CD8 T 细胞产品的可行性，为移植后同种异体免疫细胞治疗或作为现成产品提供了一种选择，以预防复发并改善 AML 患儿的临床结局。
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