Methods For Positive And Negative Selection Of Hematopoietic Progenitor Cells

点击翻译按钮获取中文摘要

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.