Luo Yan, Qie Yaqing, Gadd Martha E, Hundal Tanya, Brim Andrew D, Rosario Isas K Vazquez, Qin Hong
Regenerative Immunotherapy and CAR-T Translational Research Program, Mayo Clinic, Jacksonville, Florida, USA; Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA.
Regenerative Immunotherapy and CAR-T Translational Research Program, Mayo Clinic, Jacksonville, Florida, USA.
Cytotherapy. 2025 Apr 18. doi: 10.1016/j.jcyt.2025.04.068.
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy is an adoptive immunotherapy in which T cells are isolated from the patient and genetically modified ex vivo to express a CAR, thus enabling the CAR T-cells to target tumor cells. Currently, six Food and Drug Administration-approved CAR T-cell therapies target either CD19 for relapsed/refractory (R/R) B cell malignancies or B cell maturation antigen for R/R multiple myeloma. The success of these CAR T-cells has positioned this therapy as an important option for treating hematological malignancies and has also spurred efforts to use CAR T-cells to target solid tumors and to target nonmalignant B cell pathologies. OBJECTIVE: The final functionality of CAR T-cells is influenced by the initial T cell subpopulations that were isolated, and naïve and memory phenotypes have been shown to yield superior final products. Our goal is to refine the T cell isolation method to enrich a naïve/memory T cell population for CAR T-cell production, while excluding cell subsets that could impair CAR T-cell product performance. EXPERIMENTAL DESIGN: Healthy donor PBMCs were subjected to magnetic cell separation to deplete CD14 cells, followed by a positive selection of CD127 cells. The enriched CD14CD127 T cell population was characterized prior to CAR T-cell production. For comparison, we also generated CAR T-cells using CD14CD25CD62L T cells in an established method. Both CAR T-cell products were evaluated for production quality and compared in a series of activity assays. RESULTS: A new isolation method was applied to healthy donor PBMCs to enrich a CD14CD127 T cell population. With this approach, B cell populations and monocytes were largely excluded from the final cell population, and extremely low percentages of CD4+FoxP3+ regulatory T cells (Tregs) were detected. The enriched CD14CD127 T cell population was largely comprised of naïve, stem-like central memory, and central memory T cells. A BAFF-R targeted CAR (MC10029) was transfected into both CD14CD25CD62L T cells and CD14CD127 T cells from the same donor. Both CAR T-cell products exhibited similar cytotoxic performance. CONCLUSIONS: We developed a new two-step isolation method for enriching CD14CD127 T cells from PBMCs. This method effectively excluded unwanted B cells, yet maintained the naïve, stem-like central memory and central memory T cells that can produce functional CAR T-cells.
背景：嵌合抗原受体（CAR）T细胞疗法是一种过继性免疫疗法，即从患者体内分离T细胞，并在体外进行基因改造以表达CAR，从而使CAR T细胞能够靶向肿瘤细胞。目前，美国食品药品监督管理局批准的六种CAR T细胞疗法，要么靶向复发/难治性（R/R）B细胞恶性肿瘤的CD19，要么靶向R/R多发性骨髓瘤的B细胞成熟抗原。这些CAR T细胞的成功使这种疗法成为治疗血液系统恶性肿瘤的重要选择，也激发了人们利用CAR T细胞靶向实体瘤和非恶性B细胞疾病的努力。 目的：CAR T细胞的最终功能受最初分离的T细胞亚群影响，已证明原始和记忆表型能产生更优的最终产物。我们的目标是优化T细胞分离方法，以富集用于CAR T细胞生产的原始/记忆T细胞群体，同时排除可能损害CAR T细胞产物性能的细胞亚群。 实验设计：对健康供体的外周血单核细胞（PBMC）进行磁珠细胞分选以去除CD14细胞，随后对CD127细胞进行阳性分选。在生产CAR T细胞之前，对富集的CD14⁻CD127⁺ T细胞群体进行表征。为作比较，我们还用已确立的方法使用CD14⁻CD25⁻CD62L⁺ T细胞生成CAR T细胞。对两种CAR T细胞产物的生产质量进行评估，并在一系列活性测定中进行比较。 结果：一种新的分离方法应用于健康供体的PBMC，以富集CD14⁻CD127⁺ T细胞群体。通过这种方法，B细胞群体和单核细胞在很大程度上被排除在最终细胞群体之外，并且检测到极低百分比的CD4⁺FoxP3⁺调节性T细胞（Tregs）。富集的CD14⁻CD127⁺ T细胞群体主要由原始、干细胞样中央记忆和中央记忆T细胞组成。将靶向BAFF-R的CAR（MC10029）转染到来自同一供体的CD14⁻CD25⁻CD62L⁺ T细胞和CD14⁻CD127⁺ T细胞中。两种CAR T细胞产物均表现出相似的细胞毒性性能。 结论：我们开发了一种新的两步分离方法，用于从PBMC中富集CD14⁻CD127⁺ T细胞。该方法有效排除了不需要的B细胞，同时保留了能够产生功能性CAR T细胞的原始、干细胞样中央记忆和中央记忆T细胞。
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