A Rapid, High Performance, Cost-Efficient Clinical Scale Separator for CD3+ cells

 描述(由申请人提供)：过继细胞免疫的概念是由A.V.Mitchison1在60多年前首次提出和报道的。从那时起，人类非凡的努力和聪明才智导致了对分子生物学、免疫系统、遗传学、基因疗法和可怕的癌症的基本理解。这使得令人难以置信的工具得以创造--这在一代人之前是不可想象的--将医学置于癌症治疗的门槛上。“工具箱”的一部分是从患者/捐赠者身上分离出免疫细胞亚群，并提纯转基因细胞。目前，CD3+细胞是这种分离的主要目标。考虑到这种治疗所需的费用，具有成本效益/效率的分离至关重要。这项建议的目标是测试临床规模免疫磁性细胞分离系统的可行性，该系统具有比Miltenyi Biotec系统和许多中心使用的其他临时系统更快(3倍)、更便宜(2倍)和更好(产量和纯度)分离CD3+细胞的潜力。这一建议是基于我们的磁性纳米颗粒赋予细胞分离的非凡好处。这些磁流体是包裹着修饰蛋白质的磁铁矿晶体的晶体准球形核心，常见的捕捉剂(链霉亲和素、抗体)或单抗(直接偶联物)被偶联到修饰的蛋白质上。它们很稳定，已经投入商业使用超过10年，是FDA批准的CellSearch(R)循环肿瘤细胞浓缩计划的关键组成部分。优点：(A)扩散时的快速结合动力学(B)非凡的表面积；因此，靶向的质量输入低(C)现有的大多数磁性超顺磁性材料(大于95%的磁性超顺磁性材料-最具磁性的超顺磁性材料)(D)易于GMP制造和过滤灭菌(E)可从开放容器(试管、流动室/封闭容器与需要超高梯度磁力的Miltenyi容器、滚珠轴承填充、昂贵且低效的柱)中分离。通过内部设计的磁性设备，磁流体可以收集单层或多层的细胞，这些细胞很容易回收。与大型动态磁头不同，磁流体不会损坏细胞，不会扰乱细胞表面，也不会影响细胞活力。这项工作得益于三年来在改进我们的磁流体的磁性和表面化学方面进行的广泛研发，从而减少了材料的使用，显著降低了非特定结合，提高了纯度，并因其磁性而在收集室设计中获得了更大的自由度。从细胞分离方面的基本发现来看，使用非常少的单抗，以1010细胞起始产品的30%为目标，使用30微克单抗是可行的(而不是1000微克 相互竞争的系统)，并且只使用4毫克的第五代磁流体。而且整个分离可以在不到30分钟的时间内完成。我们的技能组合/经验：Pi创立并管理了CellSearch(R)公司，设计了肿瘤细胞在癌症早期循环的开创性实验，招募了商业和FDA批准的系统开发团队，并开始与强生公司建立合作伙伴关系。所有这一切都始于第一阶段的SBIR。1.A.V.米奇森，J Exp Med.1955年8月1日；102(2)：157-177。[PI有幸与Av Mitchison(伦敦大学学院)密切接触和互动，当时PI是Nimr的访问科学家，Mill Hill在实验室工作 已故的布里奇特·阿斯科纳斯。我们就如何利用免疫系统攻击恶性肿瘤进行了多次讨论。]

 DESCRIPTION (provided by applicant): The notion of adoptive cellular immunity was first conceived and reported by A.V. Mitchison1 more than 60 years ago. Since then extraordinary human effort and ingenuity has resulted in fundamental understandings of molecular biology, the immune system, genetics, gene therapy and the dreaded disease cancer. That has enabled the creation of incredible tools - inconceivable a generation ago - which has put medical science on the threshold of cancer cures. One part of that 'tool box' is the isolation of subsets o immune cells from patients/donors and the purification of genetically modified cells. Currently, CD3+ cells are the prime targets of such separations. Cost effective/efficient separations are vitally important given the expense such treatments entail. The goal of this proposal is to test the feasibility of a clinical scale immuno-magnetic cell separation system that has the potential to be faster (3-fold), cheaper (2-fold) and better (yield & purity) for the isolation of CD3 + cell than the system of Miltenyi Biotec and other improvised systems in use at many centers. This proposal is based on extraordinary benefits our magnetic nanoparticles confer to cell separations. These ferrofluids are crystalline quasi-spherical 120 nm cores of magnetite crystals coated with modified protein to which common capture agents (streptavidin, antibodies) or Mabs (direct conjugates) are coupled. They are stable, have been in commercial use more than 10 years and are the key component of the FDA-approved CellSearch(r) circulating tumor cell enrichment scheme. Benefits: (a) rapid binding kinetics as they diffuse (b) extraordinary surface area; thus, low mass input for targeting (c) most magnetic superparamagnetic materials in existence (greater than 95% magnetite - most magnetic superparamagnetic material) (d) easy to GMP manufacture and filter sterilize (e) separable from open vessels (test tubes, flow through chambers/closed containers vs. Miltenyi's that require ultra-high gradient magnetic forces, ball bearing packed, expensive and inefficient columns. With in-house designed magnetic devices, ferrofluids can collect cells in monolayers or multilayers that are easily retrievable. Unlike larg Dynal Dynabeads, ferrofluids do not damage cells, clutter up cell surfaces nor affect cell viability. This work benefits from 3 years of extensive R&D in improving our ferrofluids' magnetics and surface chemistries resulting in use of less material, significantly lower non-specific binding and improved purity as well as more latitude in collection chamber design because of their magnetics. From fundamental discoveries on cell separation, that use very little Mab, it is feasible to target 30% of a 1010 cell starting product with 30 ug of Mab (vs 1000 ug for competing systems) and with only 4 mg of 5th generation ferrofluid. And the entire separation can be done in less than 30 minutes. Our skill set/ experience: PI founded/managed Immunicon Corp., conceived of CellSearch(r), designed seminal experiments showing that tumor cells circulate early on in cancers, recruited the development team for commercial and FDA approved system and started the corporate partnership with Johnson & Johnson. All of that began with a Phase I SBIR. 1 . A.V. Mitchison, J Exp Med. 1955 Aug 1; 102(2): 157-177. [The PI was fortunate to have had the benefit of close contact and interactions with Av Mitchison (University College, London) while the PI was a Visiting Scientist at NIMR, Mill Hill working in the laboratory of the late Bridgette Askonas. We had many discussions on how to use the immune system to attack malignancies.]