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

摘要 利用免疫疗法对癌症的治疗前景已经从专注于以下方面的策略发展而来： 适应性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 新的免疫治疗策略只能通过定制的技术和产品来实现， 杀伤（NK）细胞，先天免疫的直接细胞毒性机制的核心-例如实现可靠的，低风险的 以及患者来源的NK细胞的低成本离体扩增，这仍然是一个强烈的未满足的需求， 免疫治疗中的弱势群体我们的建议旨在实现NK的无血清培养基补充 通过植物源成分和传统培养基补充剂的新型组合， 使用分数因子统计工具包进行配方优化的原型设计。为了实现这一目标，我们的第一阶段 该提案概述了两个具体目标-一个是优化原型无血清培养基配方， 细胞增殖和活力性能是关键的终点，两个是进一步微调原型 具有细胞毒性和与细胞凋亡和坏死相关的生物标志物终点的无血清培养基配方 NK细胞的直接细胞毒性机制，这两个特定的目标都是以统计方法为指导的， 优化.为了追求这两个特定的目标，所提出的NK细胞系是基于p53突变的。 KHYG 1 NK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，我们设想了一种新颖有效的 无血清培养基补充物，其对NK细胞生长和NK细胞表型的维持具有特异性， 功能（例如细胞毒性机制）。使能生命科学有限责任公司团队处于有利地位， 项目目标从第一阶段的具体目标开始。除了由拟议的校长领导外， 研究者（PI），Rachit Ohri博士（Enable Life Sciences的首席执行官兼创始人），具有相关专业知识， 建议的工作将得到其他团队成员的大力支持，如Donna Sonntag（来自 马萨诸塞州洛厄尔大学）和其他科学家和支持人员。埃能宝生命科学实验室位于 马萨诸塞州伍斯特市已经拥有必要的设备和仪器，即流式细胞仪，Luminex， 显微镜，BL 2（生物安全2级）生物安全柜等。此外，使能生命科学是很好的 在马萨诸塞州伍斯特周围的生态系统中根深蒂固，这对拟议的 第一阶段的目标。例如，支持性的研发和商业环境 使用先进的仪器，如多激光流式细胞仪、共聚焦荧光显微镜等。 在东北部许多校园的大学核心研究设施（CRF）。简而言之，我们 建议包括仔细结晶，相关和可实现的目标和方法，此外，我们 作为一个组织，能够很好地实现NK细胞的无血清培养基补充。

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.