PO.CL05.01 · 临床研究
通过基于细胞表面标志物的分选富集CD8+和CD4+新抗原反应性肿瘤浸润淋巴细胞
Enriching CD8+ and CD4+ neoantigen-reactive tumor infiltrating lymphocytes by cell surface marker-based sorting
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：肿瘤浸润淋巴细胞（TIL）过继细胞转移（ACT）已获FDA批准用于转移性黑色素瘤，并正在转移性上皮性癌症中进行研究。鉴定新抗原反应性TIL目前需要针对患者特异性新抗原进行耗时且资源密集的检测，只能在培养物层面进行筛选。能够分离肿瘤相关CD8⁺和CD4⁺ TIL的细胞表面标志物可消除对个性化测序和筛选的需求。
方法：对来自一处已知具有反应性的转移性胃肠道癌病灶的汇集培养物中新抗原特异性和旁观者TIL混合群体进行单细胞RNA/TCR测序，鉴定出CD103为肿瘤相关CD8⁺ TIL的首要标志物，CD31为旁观者标志物。对来自另一位患者的另一处胃肠道恶性肿瘤病灶的批量TIL进行类似分析，鉴定出PD-1和ADGRG1为CD4⁺富集标志物，CD69和KLRB1为去除标志物。检测这些标志物从汇集的上皮性癌症组织碎片（11例回顾性和5例前瞻性CD8⁺样本；10例回顾性CD4⁺样本）中富集肿瘤相关CD8⁺和CD4⁺ TIL的能力。在GREX100培养瓶中用IL-2培养21天后，根据CD103和CD31的表达对CD8⁺ TIL进行分选，根据PD1、ADGRG1、CD69和KLRB1的表达对CD4⁺ TIL进行分选。分选后的群体使用快速扩增方案（REP）进行扩增，并通过4-1BB和/或OX40表达的上调以及经ELISpot检测IFN-gamma释放来评估新抗原反应性。对各群体进行单细胞TCR测序以鉴定和确认新抗原特异性。组间比较采用非参数配对t检验。
结果：在回顾性和前瞻性样本中，CD103⁺CD31⁻ CD8⁺ TIL的平均新抗原反应性分别为19.3%和18.3%，相对于批量CD8⁺ TIL的中位富集分别为5.7倍（p = 0.0244*）和8.1倍。单细胞TCR测序证实了回顾性样本中的富集（p = 0.0312*），并验证了每例患者1-3个独特的新抗原。在10例具有CD4⁺新抗原特异性应答的患者中，CD4⁺批量TIL的平均反应性为13.8%，每例患者验证了1-5个新抗原。CD4⁺CD69⁻PD-1⁺选择使反应性中位增加2.2倍（p = 0.0195*），且相比CD4⁺批量TIL能更好地捕获新抗原特异性TCR（p = 0.0039**）。总体而言，在16例研究CD8⁺反应性的患者样本中有14例通过CD103⁺CD31⁻显示出富集，而在10例研究CD4⁺反应性的患者样本中有9例通过CD4⁺CD69⁻PD1⁺显示出富集。
结论：选择CD103⁺CD31⁻细胞可富集肿瘤相关CD8⁺ TIL，而CD4⁺CD69⁻PD-1⁺最有效地富集CD4⁺ TIL。使用这些标志物进行临床规模的分选可在无需患者特异性筛选的情况下生成新抗原富集的TIL，从而扩大对疾病进展更快患者的治疗可行性。
查看英文原文 English abstract
Introduction: Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) is FDA-approved for metastatic melanoma and is under investigation for metastatic epithelial cancers. Identifying neoantigen-reactive TIL currently requires time- and resource-intensive testing against patient-specific neoantigens, enabling selection only at the culture level. Cell-surface markers capable of isolating tumor-relevant CD8⁺ and CD4⁺ TIL could eliminate the need for personalized sequencing and screening.
Methods: Single-cell RNA/TCR sequencing of mixed population of neoantigen-specific and bystander TIL derived from pooled cultures from a metastatic GI cancer lesion with known reactivity identified CD103 as the top marker of tumor-relevant CD8⁺ TIL and CD31 as a bystander marker. Similar analysis of bulk TIL from another GI malignancy lesion derived from a different patient identified PD-1 and ADGRG1 as CD4⁺ enrichment markers and CD69 and KLRB1 as depletion markers. Markers were tested for their ability to enrich tumor-relevant CD8⁺ and CD4⁺ TIL from pooled epithelial cancer fragments (11 retrospective and 5 prospective CD8⁺ samples; 10 retrospective CD4⁺ samples). Following culture in IL-2 in GREX100 flasks for 21 days, TIL were sorted based on expression of CD103 and CD31 for the CD8⁺ TIL and PD1, ADGRG1, CD69 and KLRB1 for the CD4⁺ TIL. Sorted populations were expanded using rapid expansion protocol (REP) and evaluated for neoantigen reactivity by upregulation of 4-1BB and/or OX40 expression and IFN-gamma release via ELISpot. Single-cell TCR sequencing of populations was done to identify and confirm neoantigen specificity. Group comparisons were performed using non-parametric paired t-tests.
Results: Across retrospective and prospective samples, CD103⁺CD31⁻ CD8⁺ TIL showed mean neoantigen reactivity of 19.3% and 18.3% and median enrichment of 5.7x (p = 0.0244*) and 8.1x over bulk CD8⁺ TIL. Single-cell TCR sequencing confirmed enrichment in retrospective samples (p = 0.0312*) and validated 1-3 unique neoantigens per patient. Among 10 patients with CD4⁺ neoantigen-specific responses, CD4⁺ bulk TIL showed a mean reactivity of 13.8%, with 1-5 neoantigens validated per patient. CD4⁺CD69⁻PD-1⁺ selection produced a 2.2x median increase in reactivity (p = 0.0195*) and superior capture of neoantigen-specific TCRs vs CD4⁺ Bulk (p = 0.0039**). Overall 14 of 16 patient samples studied for CD8⁺ reactivity showed enrichment by CD103⁺CD31⁻ while 9 of 10 patient samples studied for CD4⁺ reactivity showed enrichment with CD4⁺ CD69⁻PD1⁺.
Conclusion: Selection of CD103⁺CD31⁻ cells enriches tumor-relevant CD8⁺ TIL, while CD4⁺CD69⁻PD-1⁺ most effectively enriches CD4⁺ TIL. Clinical-scale sorting using these markers could generate neoantigen-enriched TIL without patient-specific screening, expanding treatment feasibility for patients with more rapidly progressing disease.
利益披露 Disclosure
L. M. Kenney, None..
N. Ratnam, None..
A. A. Hakim, None..
J. Rault-Wang, None..
S. A. Rosenberg, None..
F. J. Lowery, None.