Methods For Positive And Negative Selection Of Hematopoietic Progenitor Cells

用于淋巴造血细胞的阳性和阴性选择的自动化封闭系统的临床前和临床研究已经与生物技术公司合作完成，这些公司已经开发了可能应用于临床细胞疗法的系统：CellPro T细胞耗竭系统：对这种两步阳性（CD 34）和阴性（CD 2）选择系统的临床评价，该系统使用免疫吸收方法，于1998年8月完成。本研究将24名同种异体供体随机分为新鲜和混合处理干细胞单采产品组。结果表明，两个研究组在处理和临床结局方面具有等效性，因此出于实际和经济原因（处理时间更短，与使用一个昂贵系统相比，使用两个昂贵系统相关的成本更低），使用了合并处理方法。该系统不再临床可用。2001年10月发表了比较该系统与Nexell Isolex系统结果的手稿（中村等人，Br J Haematol）。Nexell公司Isolex：完成了自动Isolex 300 i用于造血祖细胞免疫磁性选择的研究。在过去3年中，已完成了100多个2.0版的筛选程序（仅阳性筛选或阳性/阴性结合筛选），并完成了100多个2.5版的筛选程序。旨在实现外周血干细胞产物的最大T细胞耗竭的组合阳性+阴性选择的研究已经导致将该方法并入几种同种异体移植方案中。2.5版联合阳性/阴性程序的结果显示，平均CD 3 + T细胞消耗为5 log，平均CD 34+细胞回收率为60%。不同T细胞抗体（单独CD 2 vs. CD 4 + CD 8 vs. CD 2 + CD 6 + CD 7）的评价证明了联合阳性/阴性方法的等效性。该方法将继续用于临床试验。Miltenyi CliniMacs/CD 34阳性选择：在2000财年，我们使用该系统对正常供体动员的PBSC进行了阳性选择的临床前研究。平均CD 34+细胞回收率为55%，平均CD 3+细胞消耗为5 log。该系统可能会纳入未来的临床试验。Miltenyi CliniMacs/AC 133阳性选择：2002财年，我们与NHBLI/心脏病学分支合作，启动了一项临床前研究，选择AC 133阳性的外周血细胞，AC 133是一种更新的祖细胞标记物，包括成血管细胞谱系。到目前为止，已经完成了两个选择程序。这将持续到2003财政年度，并将作为离体产生的成血管细胞用于治疗冠状动脉和心肌疾病的临床试验的基础。 在2003财年，对Miltenyi CliniMacs系统进行了额外的AC 133选择研究，并计划在下一财年扩展这些研究，以支持心脏病学细胞治疗计划。在2006财年，逐步淘汰使用Isolex 300 i进行免疫磁性选择以富集CD 34，同时使用Miltenyi CliniMACs仪器和微珠。CliniMACs CD 34富集程序允许处理更高的TNC/处理产品和更大的CD 3细胞对数消耗。此外，Miltenyi生产的微珠对其他抗原具有特异性，因此已对其他富集和消除程序进行了临床试验评价。一个实例是CD 3消耗，随后是CD 56富集以分离用于离体培养扩增的自然杀伤（NK）细胞。

Preclinical and clinical studies of automated closed systems for positive and negative selection of lymphohematopoietic cells have been done in collaboration with biotechnology firms which have developed systems for potential application to clinical cellular therapies: CellPro T-cell Depletion System: A clinical evaluation of this two-step positive (CD34) and negative (CD2) selection system, which uses an immunoabsorption approach, was completed in August 1998. This study randomized 24 allogeneic donors to fresh versus pooled processing of stem cell apheresis products. Results demonstrated equivalence between the two study arms in processing and clinical outcomes, so the pooled processing approach was used for practical and economic reasons (less processing time, lower costs associated with use of one expensive system versus two). This system is no longer clinically available. A manuscript comparing results of this system with the Nexell Isolex system was published in October 2001 (Nakamura et al. Br J Haematol). Nexell, Inc. Isolex: Studies of the automated Isolex 300i for immunomagnetic selection of hematopoietic progenitor cells were completed. Over 100 selection procedures on version 2.0 (either positive only or combined positive/negative selection) have been completed, and over 100 selection procedures on version 2.5 were completed over the past 3 years. Studies of combined positive + negative selection aimed to achieve maximum T-cell depletion of peripheral blood stem cell products have led to incorporation of this method into several allogeneic transplantation protocols. Results on the version 2.5 combined positive/negative procedure show a mean CD3+ T-cell depletion of 5 logs, with mean CD34+ cell recovery of 60 percent. Evaluation of different T-cell antibodies (CD2 alone vs. CD4+CD8 vs. CD2+CD6+CD7) demonstrated equivalence in the combined positive/negative method. This method will continue to be used in clinical trials. Miltenyi CliniMacs/CD34 positive selection: In FY 2000, we performed a preclinical study of positive selection of normal donor mobilized PBSC using this system. Mean CD34+ cell recovery of 55 percent and a mean CD3+ cell depletion of 5 logs. This system may be incorporated into future clinical trials. Miltenyi CliniMacs/AC133 positive selection: In FY 2002, we initiated a preclinical study, in collaboration with NHBLI/Cardiology Branch, on selection of peripheral blood cells positive for AC133, a newer marker of progenitor cells that includes the angioblastic lineage. Two selection procedures have been done to date. This will be continued into FY2003, and will serve as the foundation for clinical trials of ex vivo generated angioblasts for treatment of coronary artery and myocardial disease. In FY2003, additional AC133 selection studies were performed on the Miltenyi CliniMacs system and plans were made to expand these studies in the coming fiscal year to support cardiology cell therapy initiatives. During FY2006, use of Isolex 300i for immunomagnetic selection for CD34 enrichment was gradually phased out while use of the Miltenyi CliniMACs instrument and microbeads were implemented. The CliniMACs CD34 enrichment procedure allowed for processing higher TNC per product processed and greater log depletion of CD3 cells. Also, Miltenyi manufactured microbeads with specificities for other antigens such that other enrichments and depletion procedures have been evaluated for clinical trial. One example is the CD3 depletion followed by CD56 enrichment to isolate natural killer (NK) cells for ex vivo culture expansion.