Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.临床细胞制剂普遍面临的一项核心挑战是纯度不足，这会导致细胞制品的质量与效价出现显著波动，且可能因存在非目标污染物而引发不良副作用。采用阳性选择技术可最优地获得高纯度的细胞群。然而在多数实际场景中，靶细胞群需通过多标志物组合与其他细胞实现分离，这一目标仍难以达成——对于临床细胞制剂而言尤为如此。为此，我们研发了低亲和力抗体衍生Fab片段（Fab-fragment）：该片段经链霉亲和素标签（Strep-tag）与链霉亲和素结合蛋白（Strep-Tactin）多聚化后，可展现出与亲本抗体一致的染色特性，且后续可从靶细胞群中完全去除。这类试剂几乎可针对任意抗原进行制备，并可通过顺磁磁珠开展连续阳性富集步骤。目前，我们已建立了针对两类临床相关细胞群的多参数富集首批方案：分别为CD4高表达/CD25高表达/CD45RA高表达的“调节性T细胞”，以及CD8高表达/CD62L高表达/CD45RA阴性的“中枢记忆T细胞”，以此测定该新型技术的质量与效能参数。该技术在临床细胞分选及基础研究领域均具备广泛的应用潜力。