Background/Objectives: The physiological activation of cytotoxic CD8posT cells (CTLs) relies on the engagement of the TCR/CD3 complex with cognate peptide-HLA class I (pHLA-I) on target cells, triggering cell lysis with appropriate cytokine release and minimized off-target toxicity. In contrast, current immunotherapies for CD19-expressing hematological malignancies, such as chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs),bypass TCR/pHLA interactions, resulting in CTL hyperactivation and excessive cytokine release, which frequently cause severe immune-related adverse events (irAEs). Thus, there is a pressing need for T cell-based therapies that preserve physiological activation while maintaining antitumor efficacy.Methods: To address this, we developed CD19-ReTARGTPR, a novel fusion protein consisting of the immunodominant cytomegalovirus (CMV) pp65-derived peptide TPRVTGGAM (TPR) covalently presented by a soluble HLA-B*07:02/β2-microglobulin complex fused to a high-affinity CD19-targeting Fab antibody fragment. The treatment of CD19-expressing cancer cells with CD19-ReTARGTPRmakes them recognizable for pre-existing anti-CMVpp65CTLs via physiological TCR-pHLA engagement.Results: Our preclinical data demonstrate that CD19-ReTARGTPRefficiently redirects anti-CMV CTLs to eliminate CD19-expressing cancer cells, including both established cell lines and primary chronic lymphocytic leukemia (CLL) cells. Unlike CD19-directed CAR T cells or the CD19/CD3 BiTE blinatumomab, CD19-ReTARGTPRmediatedrobustcytotoxic activity withouttriggeringsupraphysiological cytokinerelease. Importantly, this approach retained efficacy even against cancer cells with low CD19 expression.Conclusions: In summary, we provide a robust proof-of-concept study and show that CD19-ReTARGTPRoffers a promising alternative strategy for T cell redirection, enabling the selective and effective killing of CD19-expressing malignancies while minimizing cytokine-driven toxicities through physiological CTL activation pathways.
背景/目的：细胞毒性CD8阳性T细胞（CTLs）的生理性激活依赖于TCR/CD3复合物与靶细胞上同源肽-HLA I类分子（pHLA-I）的结合，从而触发细胞裂解、适当的细胞因子释放并最大限度地减少脱靶毒性。相比之下，目前针对表达CD19的血液系统恶性肿瘤的免疫疗法，如嵌合抗原受体（CAR）T细胞和双特异性T细胞衔接器（BiTEs），绕过了TCR/pHLA相互作用，导致CTL过度激活和细胞因子过度释放，这常常引发严重的免疫相关不良事件（irAEs）。因此，迫切需要一种既能保持生理性激活又能维持抗肿瘤效果的T细胞疗法。 方法：为此，我们开发了CD19-ReTARGTPR，这是一种新型融合蛋白，由免疫显性巨细胞病毒（CMV）pp65衍生肽TPRVTGGAM（TPR）共价呈递于可溶性HLA-B*07:02/β2-微球蛋白复合物上，并与高亲和力的CD19靶向Fab抗体片段融合而成。用CD19-ReTARGTPR处理表达CD19的癌细胞，可通过生理性的TCR-pHLA相互作用，使它们能够被预先存在的抗CMV pp65 CTLs识别。 结果：我们的临床前数据表明，CD19-ReTARGTPR能有效重定向抗CMV CTLs以消除表达CD19的癌细胞，包括已建立的细胞系和原发性慢性淋巴细胞白血病（CLL）细胞。与CD19导向的CAR T细胞或CD19/CD3双特异性T细胞衔接器博纳吐单抗不同，CD19-ReTARGTPR介导了强大的细胞毒活性，而不会触发超生理水平的细胞因子释放。重要的是，即使针对CD19低表达的癌细胞，该方法仍能保持疗效。 结论：总之，我们提供了一项强有力的概念验证研究，并表明CD19-ReTARGTPR为T细胞重定向提供了一种有前景的替代策略，能够通过生理性CTL激活途径，选择性地有效杀伤表达CD19的恶性肿瘤，同时最大限度地减少细胞因子驱动的毒性。
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