Translational Sciences and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, USA
Department of Medicine, University of Washington, Seattle, Washington, USA.
J Immunother Cancer. 2023 Dec 12;11(12):e007490. doi: 10.1136/jitc-2023-007490.
Myelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome genes, including , are attractive therapeutic targets as they are prevalent in MDS and sAML, arise early in neoplastic cells, and are generally absent from normal cells, including normal hematopoietic cells. MDS and sAML are susceptible to T cell-mediated killing, and thus engineered T-cell immunotherapies hold promise for their treatment. We hypothesized that targeting spliceosome mutation-derived neoantigens with transgenic T-cell receptor (TCR) T cells would selectively eradicate malignant cells in MDS and sAML.
We identified candidate neoantigen epitopes from recurrent protein-coding mutations in the spliceosome genes and using a multistep in silico process. Candidate epitopes predicted to bind human leukocyte antigen (HLA) class I, be processed and presented from the parent protein, and not to be subject to tolerance then underwent in vitro immunogenicity screening. CD8 T cells recognizing immunogenic neoantigen epitopes were evaluated in in vitro assays to assess functional avidity, confirm the predicted HLA restriction, the potential for recognition of similar peptides, and the ability to kill neoplastic cells in an antigen-specific manner. Neoantigen-specific TCR were sequenced, cloned into lentiviral vectors, and transduced into third-party T cells after knock-out of endogenous TCR, then tested in vitro for specificity and ability to kill neoplastic myeloid cells presenting the neoantigen. The efficacy of neoantigen-specific T cells was evaluated in vivo in a murine cell line-derived xenograft model.
We identified two neoantigens created from a recurrent mutation in , isolated CD8 T cells specific for the neoantigens, and demonstrated that transferring their TCR to third-party CD8 T cells is feasible and confers specificity for the U2AF1 neoantigens. Finally, we showed that these neoantigen-specific TCR-T cells do not recognize normal hematopoietic cells but efficiently kill malignant myeloid cells bearing the specific U2AF1 mutation, including primary cells, in vitro and in vivo.
These data serve as proof-of-concept for developing precision medicine approaches that use neoantigen-directed T-cell receptor-transduced T cells to treat MDS and sAML.
骨髓增生异常综合征（MDS）源自造血干细胞和祖细胞中获得的体细胞突变，导致血细胞减少，并促使向继发性急性髓系白血病（sAML）转化。剪接体基因中的复发性突变，包括 ，是有吸引力的治疗靶点，因为它们在 MDS 和 sAML 中普遍存在，在肿瘤细胞早期出现，并且通常不存在于正常细胞中，包括正常造血细胞。MDS 和 sAML 易受 T 细胞介导的杀伤，因此，工程化 T 细胞免疫疗法有望用于治疗这些疾病。我们假设，用转基因 T 细胞受体（TCR）T 细胞靶向剪接体突变衍生的新抗原，将选择性地消除 MDS 和 sAML 中的恶性细胞。
我们使用多步计算机算法从剪接体基因 和 中的复发性蛋白编码突变中鉴定候选新抗原表位。预测与人类白细胞抗原（HLA）I 类结合、由亲本蛋白加工和呈递、不受耐受影响的候选表位，然后进行体外免疫原性筛选。在体外测定中评估识别免疫原性新抗原表位的 CD8 T 细胞，以评估功能亲和力、确认预测的 HLA 限制、识别类似肽的潜力以及以抗原特异性方式杀伤肿瘤细胞的能力。对新抗原特异性 TCR 进行测序，克隆到慢病毒载体中，在敲除内源性 TCR 后转导到第三方 T 细胞中，然后在体外测试其特异性和杀伤呈递新抗原的肿瘤性髓系细胞的能力。在小鼠细胞系衍生的异种移植模型中评估新抗原特异性 T 细胞的疗效。
我们鉴定了一个来自 的复发性突变所产生的两个新抗原，分离出针对这些新抗原的特异性 CD8 T 细胞，并证明将其 TCR 转导到第三方 CD8 T 细胞是可行的，并赋予对 U2AF1 新抗原的特异性。最后，我们表明，这些新抗原特异性 TCR-T 细胞不识别正常造血细胞，但能有效杀伤体外和体内携带特定 U2AF1 突变的恶性髓系细胞，包括原代细胞。
这些数据为开发使用新抗原导向的 T 细胞受体转导 T 细胞治疗 MDS 和 sAML 的精准医学方法提供了概念验证。
