Methods For Positive And Negative Selection Of Hematopoietic Progenitor Cells

与生物技术公司合作，对淋巴造血细胞阳性和阴性自动封闭选择系统进行了临床前和临床研究，这些公司开发了潜在应用于临床细胞治疗的系统：CellPro T细胞耗竭系统：使用免疫吸收方法的这种两步阳性(CD34)和阴性(CD2)选择系统的临床评估于1998年8月完成。这项研究随机选择了24名同种异体捐献者，分别使用新鲜和混合处理的干细胞分离产品。结果表明，两个研究部门在处理和临床结果方面是相同的，因此使用联合处理方法是出于实际和经济原因(与使用两个昂贵的系统相比，使用一个昂贵的系统所需的处理时间更短，成本更低)。这个系统不再在临床上可用。2001年10月出版了将该系统与Nexell Isolex系统的结果进行比较的手稿(Nakamura等人)。BrJ Haematol)。Nexell，Inc.Isolex：用于造血祖细胞免疫磁性选择的自动化Isolex 300i的研究已经完成。已经完成了关于2.0版的100多个选择程序(纯正面选择或正面/负面组合选择)，并在过去3年中完成了关于2.5版的100多个选择程序。旨在最大限度地消耗外周血干细胞产品的T细胞的联合阳性和阴性选择的研究已经导致将这种方法纳入到几种异基因移植方案中。在2.5版联合阳性/阴性程序上的结果显示，CD3+T细胞平均耗尽5个对数，平均CD34+细胞回收率为60%。对不同T细胞抗体(CD2单独与CD4+CD8与CD2+CD6+CD7)的评估在联合阳性/阴性方法中显示出等效性。这种方法将继续用于临床试验。Miltenyi CliniMacs/CD34阳性选择：2000财年，我们利用该系统进行了一项正常供者动员的PBSC阳性选择的临床前研究。平均CD34+细胞回收率为55%，平均CD3+细胞耗竭为5个对数。该系统可能会被纳入未来的临床试验。Miltenyi CliniMacs/AC133阳性选择：在2002财年，我们与NHBLI/心脏病学分会合作，启动了一项临床前研究，目的是选择AC133阳性的外周血细胞，AC133是包括血管母细胞系在内的祖细胞的较新标记物。到目前为止，已经完成了两个遴选程序。这将持续到2003财年，并将作为体外生成的血管母细胞用于治疗冠状动脉和心肌疾病的临床试验的基础。2003财年，在Miltenyi CliniMacs系统上进行了额外的AC133选择研究，并计划在下一财年扩大这些研究，以支持心脏病细胞治疗的倡议。在2006财年期间，在使用Miltenyi CliniMACs仪器和微珠的同时，逐步淘汰了使用Isolex 300i进行CD34浓缩的免疫磁性选择。CliniMACs CD34浓缩程序允许处理每个处理的产品更高的TNC和更大的CD3细胞对数消耗。此外，Miltenyi还制造了对其他抗原具有特异性的微珠，以便对其他浓缩和去除过程进行临床试验评估。一个例子是CD3耗尽，然后CD56浓缩以分离自然杀伤(NK)细胞用于体外培养扩增。

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.