High Performance Magnetic Cell Sorting

描述(由申请人提供)：一个越来越受关注的领域，也是潜在的重要临床应用，是利用相关捐赠者为高危和难治性血液系统恶性肿瘤进行不匹配的干细胞移植。这类患者目前正在接受使用人类白细胞抗原相合的同胞或无关捐赠者干细胞的移植。使用来自兄弟姐妹、父母或子女的单倍型不匹配的捐赠者，将允许超过90%-95%的候选患者获得合适的捐赠者。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细胞耗尽(&gt；5logs)来消除。虽然T细胞耗尽会增加移植排斥反应的风险，但这可以通过使用非常大剂量的干细胞来克服。因此，人们希望有一种系统，其中非常大量的干细胞可以被有效地处理以耗尽T细胞，并且这些干细胞的损失最小。 人们越来越感兴趣的第二个领域，以及潜在的重要临床应用，是分离自然杀伤(NK)细胞用于免疫治疗。NK细胞是天然免疫系统中的重要细胞，不参与特异性抗原识别。这些细胞在防御感染方面很重要，但也有强大的抗肿瘤作用。目前，利用自体和同种异体激活的NK细胞进行肿瘤免疫治疗的兴趣日益浓厚。特别是，使用与受体的HLA-C等位基因不匹配的同种异体NK细胞在体外可以发挥非常强大的抗白血病作用。在骨髓移植中，NK细胞的输注也可能有助于干细胞的植入。 该项目将专注于开发/应用一种高通量、流经免疫磁性细胞分离系统，目前正在开发中，用于临床规模的T细胞去除和NK分离。虽然已经开发了几种系统，但目前还没有一种系统被批准用于这些适应症，而且它们的性能通常是次优的。关于T细胞耗尽过程中干细胞的恢复，需要显著增加干细胞恢复，从报道的平均值41%提高到80%以上，以促进在不匹配的移植中进行大剂量的CD34细胞治疗。临床规模的NK细胞分离技术的研究较少，目前的方法由于损失了50%以上的NK细胞而表现不佳。因此，我们的具体目标是： 1.在T细胞耗竭方面表现出显著的优越性能，目标是实现5log10的T细胞耗竭，在大于或等于80%的来自正常供者的临床采集样本中，CD34+细胞的回收率为90%。 2.显示出显著优越的NK细胞分离性能，目的是分离CD56+CD3-NK细胞，回收率为90%，CD3+细胞污染率为1×10/5CD3+细胞污染率大于或等于90%。

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.