A Novel Serum Alternative for Ex-vivo Expansion of Natural Killer Cells for Immunotherapy

用于免疫治疗的自然杀伤细胞离体扩增的新型血清替代品

• 批准号: 10011207
• 负责人: Rachit Ohri
• 金额: $ 22.4万
• 依托单位: ENABLE LIFE SCIENCES LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011207
• 关键词: A549; ANXA5 gene; Achievement; Address; Antigens; Apoptosis; Biological; Biological Markers; Biological Sciences; Businesses; Cell Culture Techniques; Cell Line; Cell Maintenance; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cellular immunotherapy; Chemicals; Clinical; Coculture Techniques; Computers and Advanced Instrumentation; Crystallization; Culture Media; Custom; Cyclic GMP; Data; Development; Disease; Ecosystem; Environment; Enzyme-Linked Immunosorbent Assay; Equipment; Flow Cytometry; Fluorescence Microscopy; Formulation; Garlic Extract; Goals; Granzyme; Growth Factor; Human; Human body; Immune; Immunotherapeutic agent; Immunotherapy; Interferon Type II; K562 Cells; Lasers; Lead; Malignant Neoplasms; Malignant neoplasm of lung; Massachusetts; Mathematics; Methodology; Methods; Microscope; Mutation; NK Cell Activation; Natural Immunity; Natural Killer Cell toxicity; Natural Killer Cells; Natural Products; Necrosis; Nutrient; Organic Synthesis; Patients; Performance; Phase; Phenotype; Plant Extracts; Plants; Polysaccharides; Positioning Attribute; Principal Investigator; Propidium Diiodide; Recipe; Resveratrol; Risk; Safety; Scientist; Serum; Serum-Free Culture Media; Site; Small Business Innovation Research Grant; Source; Standardization; TNF gene; TP53 gene; Technology; Therapeutic; Time; Universities; Vitamins; adaptive immunity; base; cancer cell; cell growth; checkpoint inhibition; chemical standard; chimeric antigen receptor T cells; cost; cytokine; cytotoxic; cytotoxicity; design; experimental study; fetal bovine serum; innovation; instrumentation; member; novel; perforin; programs; prototype; research and development; research facility; sound; synergism; tool

Abstract Harnessing the therapeutic promise of immunotherapy against cancer has evolved from strategies focused on the antigen-recognition mechanisms of adaptive immunity (e.g. checkpoint inhibition and CAR-T cells) to strategies leveraging direct cytotoxicity mechanisms of innate immunity. The clinical impact of these exciting new immunotherapy strategies can be realized only with customized technologies and products for Natural Killer (NK) cells, central to direct cytotoxicity mechanisms of innate immunity - e.g. achieving reliable, low-risk and low-cost ​ex vivo expansion of patient-derived NK cells, which remains a strong unmet need and an underserved niche in immunotherapy. Our proposal aims to achieve a serum-free media supplement for NK cells through a novel combination of plant-derived ingredients and traditional media supplements, guided in prototyping with a fractional-factorial statistical toolkit for recipe optimization. To achieve this goal, our Phase-I proposal outlines two specific aims - one to optimize the prototype serum-free media recipe with cell-proliferation and viability performance being key end-points, and two to further fine-tune the prototype serum-free media recipe with cytotoxicity and biomarker end-points pertaining to both apoptosis and necrosis mechanisms of direct cytotoxicity by NK cells, both specific aims guided with a statistical approach for efficient optimization. For the pursuit of both these specific aims, the proposed NK cell line is the p53 mutation based KHYG1 NK cell line, followed by a confirmation of the key experimental end-points with 3 distinct batches of primary human NK cells. At the end of Phase-I, we envision a lead formulation for a novel and effective serum-free media supplement that is specific to NK cell growth and maintenance of NK cell phenotype and functions (e.g. cytotoxicity mechanisms). The Enable Life Sciences LLC team is well positioned to pursue the project goal starting with the proposed Phase-I specific aims. In addition to being led by the proposed Principal Investigator (PI), Dr. Rachit Ohri (CEO and Founder of Enable Life Sciences) who has the relevant expertise, the proposed effort will be well supported by other team members such as Donna Sonntag (MS from the University of Massachusetts, Lowell) and other scientists and support staff. The Enable Life Sciences labs in Worcester MA already houses the requisite equipment and instrumentation, namely flow cytometers, Luminex, microscopes, BL2 (biosafety level 2) biological safety cabinets etc. Moreover, Enable Life Sciences is well entrenched in an ecosystem in an around Worcester MA, which is deeply facilitative for the proposed objectives of this Phase-I proposal. These include a supportive R&D and business environment, for example access to advanced instrumentation such as multi-laser flow cytometry, confocal fluorescence microscopy etc. at University Core Research Facilities (CRFs) at numerous campus sites in the North-East. In a nutshell, our proposal includes carefully crystallized, relevant and achievable objectives and methodologies, additionally we are well-positioned as an organization to achieve the envisioned serum-free media supplement for NK cells.

摘要 利用针对癌症的免疫疗法的治疗前景已经从专注于 获得性免疫(如检查点抑制和CAR-T细胞)的抗原识别机制 利用先天免疫的直接细胞毒性机制的策略。这些激动人心的临床影响 只有通过为Natural定制的技术和产品才能实现新的免疫治疗策略 杀伤(NK)细胞，先天免疫的中心到直接的细胞毒性机制--例如，实现可靠、低风险 以及低成本的​体外扩增患者来源的NK细胞，这仍然是一个强烈的未得到满足的需求和 免疫治疗中服务不足的利基市场。我们的建议旨在实现NK的无血清媒体补充 通过植物衍生成分和传统培养基补充剂的新组合，引导细胞 使用分数阶乘统计工具包进行配方优化的原型制作。为了实现这一目标，我们的第一阶段 提案概述了两个具体目标-一个是优化原型无血清媒体配方， 细胞增殖和存活性能是关键的终点，还有两个进一步微调原型的关键 具有细胞毒性和与细胞凋亡和坏死相关的生物标记物终点的无血清培养配方 NK细胞直接细胞毒作用的机制，这两个特定的目标都是用统计学方法指导的有效率 优化。为了追求这两个特定的目标，建议的NK细胞系是以p53突变为基础的 KHYG1NK细胞系，然后用3个不同批次的 原代人类NK细胞。在第一阶段结束时，我们设想了一个新的和有效的领先配方 对NK细胞的生长和NK细胞表型的维持具有特异性的无血清补充剂 功能(例如，细胞毒性机制)。Enable Life Science LLC团队处于有利地位，可以追求 项目目标从拟议的第一阶段具体目标开始。除了由拟议的校长领导外 研究员(PI)，Rachit Ohri博士(Enable Life Science的首席执行官兼创始人)，他拥有相关专业知识， 提议的工作将得到其他团队成员的良好支持，如Donna Sonntag(来自 马萨诸塞大学洛厄尔分校)和其他科学家和支持人员。Enable Life Science实验室位于 伍斯特马萨诸塞州已经拥有必要的设备和仪器，即流式细胞仪，Luminex， 显微镜，BL2(生物安全2级)生物安全柜等。此外，Enable Life Science是很好的 在马萨诸塞州伍斯特周围的生态系统中根深蒂固，这对计划的 此第一阶段提案的目标。例如，这些包括支持性的研发和商业环境 获得先进的仪器，如多激光流式细胞仪、共聚焦荧光显微镜等。 在东北部许多校区的大学核心研究设施(CRF)。简而言之，我们的 建议包括仔细明确的、相关的和可实现的目标和方法，此外，我们 作为一个组织，我们处于有利地位，能够实现设想的NK细胞无血清介质补充剂。