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.

抽象的 利用免疫疗法对癌症的治疗承诺已从专注于 自适应免疫的抗原识别机制（例如检查点抑制和CAR-T细胞） 利用先天免疫的直接细胞毒性机制的策略。这些令人兴奋的临床影响 新的免疫疗法策略只能通过自然的定制技术和产品来实现 杀手级（NK）细胞，定于先天免疫的直接细胞毒性机制的中心 - 例如实现可靠的低风险 和患者来源的NK细胞的低成本离体扩展，这仍然是强大的未满足需求，并且 免疫疗法中服务不足的利基市场。我们的建议旨在为NK提供无血清的媒体补充 通过新颖的植物衍生成分和传统媒体补充剂的组合组合的细胞，以此为指导 具有分数因素统计工具包的原型制作，用于配方优化。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 - 一个用来优化无原型血清媒体配方 细胞增殖和生存力性能是关键终点，两个进一步调整了原型 具有细胞毒性和生物标志物终点的无血清介质配方与凋亡和坏死有关 NK细胞直接细胞毒性的机制，这两个特定目的都以统计方法为指导 优化。为了追求这两个特定目标，提出的NK细胞系是基于p53突变 khyg1 nk细胞系，随后确认关键实验终点，有3个不同的批次 原代人NK细胞。在第一阶段结束时，我们设想了一个新颖有效的铅公式 无血清培养基补充剂，特定于NK细胞的生长和NK细胞表型的维持和维持 功能（例如细胞毒性机制）。启用生命科学有限责任公司团队有能力追求 项目目标从拟议的I期特定目标开始。除了由拟议的校长领导 研究人员（PI），Rachit Ohri博士（Enable Life Sciences的首席执行官兼创始人），拥有相关专家， 拟议的努力将得到其他团队成员的支持，例如Donna Sonntag（来自The MS 马萨诸塞大学，洛厄尔大学）和其他科学家和支持人员。启用生命科学实验室 MA伍斯特已经装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）生物安全柜等。此外，启用生命科学还不错 在马萨诸塞州伍斯特周围的生态系统中根深蒂固，这对拟议的人非常友好 该阶段提案的目标。例如，其中包括支持的研发和商业环境 访问高级仪器，例如多激光流式细胞仪，共聚焦荧光显微镜等。 在东北众多校园地点的大学核心研究设施（CRFS）。简而言之 提案包括精心结晶，相关和成功的目标和方法，此外，我们 作为一个组织，可以很好地实现NK细胞的无血清培养基补充剂。