Long-Term TIL Engraftment and Clonal Expansion After Multiple TIL and Anti-PD-1 Therapy: A Five-Year Immunogenomic Case Study

Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) can achieve durable responses in metastatic melanoma and other indications, but the long-term clonal dynamics after multiple administration and synergy with checkpoint blockade are still to be explored. We present a longitudinal case study of a prostate cancer patient treated with serial TIL infusions and delayed anti-PD-1 therapy, analyzed using high-throughput T-cell receptor (TCR) sequencing from serial blood and tumor samples over five years. TIL-derived clonotypes exhibited sustained persistence and clonal expansion in blood, with peaks corresponding to clinical response. Multiple TIL administration increased the patient exposure to the therapy, improving its pharmacokinetics profile over time. Notably, anti-PD-1 therapy administered six months post-first TIL administration further expanded both previously infused and novel TIL-derived clonotypes, coinciding with durable complete response. Serial tracking revealed stable levels of TIL-derived clonotypes up to five years post-treatment, with persistent tumor infiltration. T-cell clonotype expansion was associated with decreased TCR diversity and increased frequency of hyperexpanded clones, suggesting selective amplification of tumor-reactive populations. Our findings provide mechanistic insight into the long-term persistence and reactivation of TIL-derived immunity and illustrate the potent synergy between adoptive transfer and delayed PD-1 blockade, supporting the integration of longitudinal immunogenomic monitoring in personalized immunotherapy. Overall design: Peripheral blood and tumor biopsies were collected at baseline, following each TIL infusion, and periodically up to five years. TCRß deep sequencing was performed to track TIL-derived, blood-borne, and unique clonotypes longitudinally D= day M= month W= week Y= year TIL-1-D1: sample collected one day after first infusion, TIL-2-D1: sample collected one day after second infusion, and so forth.

使用肿瘤浸润淋巴细胞（Tumor-Infiltrating Lymphocytes, TIL）的过继性细胞疗法（Adoptive Cell Therapy, ACT）可在转移性黑色素瘤及其他适应症中实现持久应答，但多次输注后的长期克隆动态以及与免疫检查点阻断的协同效应仍有待探索。我们报道了一例接受序贯TIL输注与延迟性抗PD-1治疗的前列腺癌患者的纵向病例研究，通过对五年间系列血液与肿瘤样本开展高通量T细胞受体（T-cell receptor, TCR）测序进行分析。TIL来源的克隆型在血液中表现出持续存活与克隆扩增，其扩增峰值与临床应答相对应。多次TIL输注提升了患者的治疗暴露量，随时间推移改善了其药代动力学特征。值得注意的是，首次TIL输注六个月后给予的抗PD-1治疗进一步扩增了既往输注的与新出现的TIL来源克隆型，与持久完全应答相吻合。系列追踪显示，治疗后长达五年的TIL来源克隆型水平保持稳定，且肿瘤浸润持续存在。T细胞克隆型扩增与TCR多样性降低及过度扩增克隆的频率升高相关，提示肿瘤反应性细胞群发生了选择性扩增。我们的研究结果为TIL来源免疫的长期存活与再激活提供了机制层面的见解，并阐明了过继转移与延迟PD-1阻断之间的强效协同效应，支持将纵向免疫基因组监测整合入个性化免疫治疗方案。整体实验设计：在基线期、每次TIL输注后以及长达五年的随访周期内，定期采集外周血与肿瘤活检样本。通过TCRβ深度测序，纵向追踪TIL来源、血液来源及独特克隆型。样本命名规则如下：D=日，M=月，W=周，Y=年；例如TIL-1-D1为首次输注后1天采集的样本，TIL-2-D1为第二次输注后1天采集的样本，依此类推。