Vaccine Educated T cells and a CD123 Bi-Specific T-Cell Engager for Treatment of Acute Myeloid Leukemia

Immunotherapy strategies in acute myeloid leukemia remain limited. CD123 is a promising target as it is overexpressed in myeloid blasts. This preclinical work investigates possible synergy of CD123 T cell engager and dendritic cell based vaccine, which can induce the expansion of leukemia specific T cells. Efficacy of combination therapy was evaluated in a patient-derived xenograft model and the infused T cells were later collected for single cell rnaseq to determine effects on gene expression pathways and clonality. In the present study, we demonstrated that the combination of DC/AML fusion vaccine and a CD123 T cell engager SAR440234 led to an increase in tumor specific T cell immunity and enhanced anti-leukemia effect in vitro. Furthermore, the combination treatment was shown to increase cytotoxic T cell subsets and clonotypic expansion and eradicate disease in vivo. Thus the combination of T cell engager and vaccination or adoptive T cell transfer is a novel approach that merits further investigation in clinical trials. Cell suspensions following harvesting of bone marrow and spleen tissues from individual mice were cryopreserved. NSG mice had been injected with human T cells. Upon thawing, samples underwent dead cell removal (Miltenyi MACS kit) and human CD45 enrichment (Stem Cell Technologies). Based on cell yield, samples from 4 mice per treatment group (no treatment, Tc+IgG, uTc+SAR, veTc+IgG, veTc+SAR) were chosen for single cell RNA and TCR sequencing, yielding 20 samples from bone marrow and spleen compartments each. 5 spleen or bone marrow samples were pooled together (one sample per treatment group), and 32,000-40,000 cells were loaded in one channel of a Chromium Chip K (10x Genomics) and profiled using the Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics). Full-length paired α/β TCR libraries were obtained using the Chromium Single Cell V(D)J Enrichment Human T Cell (10x Genomics). cDNA, gene expression, and TCR library quality was assessed using the DNA High Sensitivity Bioanalyzer Chip (Agilent). Gene expression or TCR libraries were pooled and sequenced on a NovaSeq 6000 sequencer (Illumina).

急性髓系白血病（acute myeloid leukemia）的免疫治疗策略仍十分有限。CD123是极具潜力的治疗靶点，因其在髓系母细胞中呈高表达。本临床前研究旨在探究CD123 T细胞衔接器（T cell engager）与树突状细胞（dendritic cell）疫苗的协同作用潜力——后者可诱导白血病特异性T细胞的扩增。联合疗法的疗效在患者来源的异种移植模型（patient-derived xenograft model）中进行评估，随后收集输注的T细胞进行单细胞RNA测序（single-cell RNA-seq），以解析其对基因表达通路及克隆性的影响。

本研究证实，树突状细胞/急性髓系白血病融合疫苗与CD123 T细胞衔接器SAR440234联合使用，可在体外增强肿瘤特异性T细胞免疫应答，并提升抗白血病效应。进一步研究表明，该联合疗法可增加细胞毒性T细胞亚群比例、促进克隆性扩增，并在体内清除白血病病灶。因此，T细胞衔接器与疫苗接种或过继性T细胞转移（adoptive T cell transfer）的联合方案是一种全新的治疗策略，值得在临床试验中开展进一步研究。

从单只小鼠的骨髓与脾脏组织中获取的细胞悬液经冷冻保存。此前已向NSG免疫缺陷小鼠输注人T细胞。样本解冻后，先后经死细胞去除试剂盒（Miltenyi MACS试剂盒）与人CD45富集试剂盒（Stem Cell Technologies）处理。根据细胞产出量，每组治疗组（未处理组、Tc+IgG组、uTc+SAR组、veTc+IgG组、veTc+SAR组）选取4只小鼠的样本，最终从骨髓与脾脏两个组织分区各获得20份样本。将5份脾脏或骨髓样本合并为1份（每个治疗组对应1份合并样本），在Chromium Chip K（10x Genomics）的单个通道中加载32000~40000个细胞，并使用Chromium Next GEM Single Cell 5' Kit v2（10x Genomics）进行建库分析。通过Chromium Single Cell V(D)J Enrichment Human T Cell Kit（10x Genomics）构建全长配对α/β T细胞受体（TCR）文库。使用DNA High Sensitivity Bioanalyzer Chip（Agilent）评估cDNA、基因表达文库及TCR文库的质量。将基因表达文库或TCR文库混合后，在NovaSeq 6000测序仪（Illumina）上完成测序。