High Performance Magnetic Cell Sorting

描述（由申请人提供）：一个越来越受关注的领域，以及潜在的重要临床用途，是使用相关供体对高风险和难治性血液恶性肿瘤进行不匹配的干细胞移植。这些患者目前使用HLA匹配的同胞或无关供体干细胞进行移植。使用来自兄弟姐妹、父母或子女的单倍型错配供体将允许超过90-95%的候选患者获得合适的供体。然而，主要的限制是移植物抗宿主病（GVHD）。GVHD几乎可以通过广泛的T细胞耗竭（>5 log）消除。虽然T细胞耗竭增加了移植排斥的风险，但这可以通过使用非常大剂量的干细胞来克服。因此，希望有一种系统，其中可以有效地处理非常大量的干细胞以耗尽T细胞，并且这些干细胞的损失最小。 第二个越来越受关注的领域和潜在的重要临床用途是分离用于免疫治疗的自然杀伤（NK）细胞。NK细胞是先天免疫系统的重要细胞，不参与特异性抗原识别。这些细胞在抵抗感染方面很重要，但也具有强大的抗肿瘤作用。目前，人们对使用自体和同种异体活化NK细胞进行癌症免疫治疗的兴趣越来越大。特别地，使用与受体的HLA-C等位基因错配的同种异体NK细胞可以在体外发挥非常有效的抗白血病作用。NK细胞的输注也可以帮助骨髓移植环境中干细胞的植入。 该项目将专注于目前正在开发的用于临床规模T细胞去除和NK分离的高通量、流通免疫磁性细胞分离系统的开发/应用。虽然已经开发了几种系统，但目前没有一种被批准用于这些适应症，并且它们的性能通常不是最佳的。关于T细胞耗竭期间干细胞的回收，需要显著增加干细胞回收，从报道的平均值41%增加到超过80%，以促进错配移植中的“大剂量”CD 34细胞治疗。对临床规模的NK细胞分离技术进行的研究较少，目前的方法由于损失超过50%的NK细胞而表现不佳。因此，我们的具体目标是： 1.证明T细胞去除的显著上级性能，目的是实现5 log 10的T细胞去除，在大于或等于80%的从正常供体获得的临床单采血液成分术样品中，CD 34+细胞的回收率>90%。 2.证明NK细胞分离的显著上级性能，目的是在大于或等于90%的外周血单采产品中分离回收率> 90%且CD 3+细胞污染<1 x 10/5的CD 56 + CD 3- NK细胞。

DESCRIPTION (provided by the applicant): An area of increasing interest, and potentially significant clinical use, is performing mismatched stem cell transplants for high-risk and refractory hematological malignancies using related donors. Such patients currently undergo transplants using HLA matched sibling or unrelated donor stem cells. Using haplotype-mismatched donors from siblings, parents or children would permit the availability of suitable donors for more than 90-95% of candidate patients. However, the main limitation is graft-versus-host disease (GVHD). GVHD can be almost eliminated by extensive T-cell depletion (>5 logs). While T cell depletion increases the risk of graft rejection, this can be overcome by the use of very large doses of stem cells. Therefore, it is desirable to have a system where very large numbers of stem cells can be efficiently processed to deplete T-cells, and with minimal loss of these stem cells. A second area of increasing interest, and potentially significant clinical use, is the isolation of Natural Killer (NK) Cells for Immunotherapy. NK cells are important cells of the innate immune system that are not involved in specific antigen recognition. These cells are important in the defense against infections, but also have potent anti-tumor effects. Currently, there is growing interest in the use of both autologous and allogeneic activated NK cells for cancer immunotherapy. In particular, the use of allogeneic NK cells mismatched for HLA-C alleles of the recipient can exert very potent anti-leukemic effect in vitro. Infusion of NK cells may also assist engraftment of stem cells in the bone marrow transplant setting. This project will focus on the development/application of a high throughput, flow through immunomagnetic cell separation system currently under development for clinical scale T-cell depletion and NK isolation. While several systems have been developed, none are currently approved for these indications, and their performance is generally suboptimal. With respect to the recovery of stem cells during T-cell depletion, a significant increase in stem cell recovery is needed, from reported mean values of 41 to over 80%, to facilitate 'mega-dose' CD34 cell therapy in mismatched transplants. Less research has been carried out for clinical scale NK cell separation technology, with current methods performing sub-optimally due to loss of over 50% of the NK cells. Specifically, therefore, our objectives are: 1.To demonstrate significantly superior performance for T-cell depletion with the aim of achieving 5 log10 depletion of T cells, with >90% recovery of CD34+ cells in greater than or equal too 80% of clinical apheresis samples obtained from normal donors. 2. To demonstrate significantly superior performance of NK cell isolation with the aim of isolating CD56+ CD3- NK cells with > 90% recovery and with <1 x 10/5 CD3+ cell contamination in greater than or equal to 90% of peripheral blood apheresis products processed.