Novel cryopreservation method for stabilization of manufactured therapeutic cells

用于稳定制造的治疗细胞的新型冷冻保存方法

• 批准号: 10272461
• 负责人: Alptekin Aksan
• 金额: $ 16.07万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10272461
• 关键词: Adoption; Adverse effects; Affect; Allogenic; Antigen Presentation; Attention; Autologous; Blood; Cancer Patient; Cell Count; Cell Survival; Cell Therapy; Cell membrane; Cell physiology; Cells; Clinic; Clinical; Consumption; Cryopreservation; Crystallization; Culture Media; Cyclic GMP; Derivation procedure; Development; Dimethyl Sulfoxide; Dose; Dry Ice; Engineering; Formulation; Freezing; Gel; Goals; Growth; Hematologic Neoplasms; Hematopoietic Neoplasms; Hydrogels; Ice; Immunotherapy; Infusion procedures; Institution; Letters; Liquid substance; MHC Class I Genes; MHC antigen; Malignant Neoplasms; Methods; Mind; Minnesota; Molecular; NK cell therapy; Natural Killer Cells; Nitrogen; Patients; Problem Solving; Process; Production; Protocols documentation; Reagent; Risk; Solid Neoplasm; Speed; Standardization; Technology; Temperature; Testing; Therapeutic; Time; Transfusion; Tumor Escape; Universities; anticancer activity; base; cancer therapy; cell growth; chimeric antigen receptor T cells; clinical practice; cost; cost effectiveness; cryogenics; cytokine; cytotoxic; cytotoxicity; extracellular; fetal bovine serum; graft vs host disease; induced pluripotent stem cell; innovation; manufacturing facility; mechanical force; melting; novel; peripheral blood; preservation; programs; response

Novel Cytocompatible Encapsulation-based Vitrification Method for Natural Killer Cell Immunotherapy Summary: Use of natural killer (NK) cells as immunotherapy against hematologic and solid tumor cancers has gained much attention in the last few years. Critical advantages of NK cells include lack of risk for transfusion- associated graft-versus-host disease and decreased likelihood of tumor escape. The fact that NK cells recognize targets without needing MHC class I allows for the use of allogeneic products. At the University of Minnesota, co-I Dr. Miller and his NK cell Program team have treated over 400 cancer patients with donor NK cells and single dosing is a major limitation to fully test their anti-cancer activity. To allow for third party, off-the- shelf large- scale manufacturing needed for multidose strategies, we must be able to freeze and thaw NK cells on demand with minimal loss of viability and function. However, current cryopreservation practice heavily relies on the use of toxic cryoprotectant agents (CPAs) such as dimethyl sulfoxide (DMSO) and freeze/thaw protocols that are not optimized for NK cells. As a result, there is a) a significant decrease in post-thaw viability of the NK cells, and b) loss of NK cell cytotoxicity and cytokine production function. PI Prof. Aksan's lab has recently developed a cytocompatible encapsulation-based vitrification method that does not require cytotoxic and mutagenic CPAs. The main goal of this proposal is development and adaptation of this novel method to vitrify and preserve expanded blood and induced pluripotent stem cell (iPSC) derived NK cells. Our method avoids detrimental intra-/extracellular ice crystallization during cooling, and re-crystallization during warming, therefore eliminating freeze/thaw and cryogenic storage-induced loss of viability and post-thaw function. To reach our main goal, we will pursue the following specific aims: Specific Aim 1: Engineer the encapsulation gel and determine the cytocompatible CPA compositions that will inhibit intra-/extracellular ice crystallization and growth, devitrification, and eutectic melting, which are detrimental. Specific Aim 2: Maximize post-thaw NK cell viability and function using optimized freeze-thaw protocols. Specific Aim 3: Verify short and medium-term storage stability of NK cells at -80oC, and in liquid nitrogen.

一种新的细胞相容性玻璃化冷冻方法 自然杀伤细胞免疫疗法 概述：使用自然杀伤（NK）细胞作为针对血液和实体瘤癌症的免疫疗法， 在过去的几年里受到了广泛的关注。NK细胞的关键优势包括没有输血风险- 相关的移植物抗宿主病和降低肿瘤逃逸的可能性。NK细胞 识别目标而不需要MHC I类允许使用同种异体产品。大学的 米勒博士和他的NK细胞项目团队已经用供体NK治疗了400多名癌症患者 细胞和单次给药是全面测试其抗癌活性的主要限制。为了让第三方，关闭- 搁置多剂量策略所需的大规模生产，我们必须能够冷冻和解冻NK细胞 以最小的生存能力和功能损失。然而，目前的冷冻保存实践严重依赖于 使用毒性冷冻保护剂（CPA），如二甲基亚砜（DMSO）和冷冻/解冻方案 不是针对NK细胞的结果，a）NK细胞的解冻后活力显著降低， 细胞，和B）NK细胞细胞毒性和细胞因子产生功能的丧失。 PI Aksan教授的实验室最近开发了一种基于细胞相容性的玻璃化方法， 不需要细胞毒性和致突变CPA。该提案的主要目标是发展和 采用这种新方法来玻璃化和保存扩增的血液和诱导多能干细胞 （iPSC）衍生的NK细胞。我们的方法避免了冷却过程中有害的细胞内/细胞外冰结晶， 和在加热过程中重结晶，因此消除了冻/融和低温储存引起的 活力和解冻后功能。为了实现我们的主要目标，我们将追求以下具体目标： 具体目标1：设计包封凝胶并确定细胞相容性CPA组合物 这将抑制内部/外部冰结晶和生长、失透和共晶熔化， 这是有害的。 具体目标2：使用优化的冻融方法最大化解冻后NK细胞活力和功能 协议. 具体目标3：验证NK细胞在-80 ℃和液体中的短期和中期储存稳定性 氮气