Developing Monoclonal Antibody Secreting Human Plasma Cells to Provide Long-Lasting EBV Immunity in Humanized Mice

摘要 单克隆抗体（mAb）疗法已被证明是多种临床疾病的有效治疗。 应用.这些治疗中的一些需要通过静脉输注重复给药， 高成本和患者依从性差。一个新的领域已经出现，寻求产生单克隆抗体分泌 浆细胞治疗剂，其可以一次施用以提供终身治疗性mAb水平， 替代疗法浆细胞具有高分泌能力，可以持续一生，并且被认为是 相对安静。基因编辑和离体B细胞分化的进展已经使得能够 产生分泌外源mAb的浆细胞。然而，目前的基因编辑策略并不完全 解释内源性抗体表达或控制外源性mAb表达为何种同种型。 此外，基因编辑的人浆细胞领域缺乏大型和小型动物模型来测试基因编辑的人浆细胞。 这些分泌mAb的浆细胞的移植和功能。 本提案的目标1将使用新的基因工程策略来产生人浆细胞治疗剂 其分泌针对爱泼斯坦巴尔病毒（EBV）的工程化IgG mAb，作为未来mAb的概念验证， 分泌细胞疗法我将描述基因编辑的原代浆细胞产生大细胞的能力， 定量的确定的mAb，并与先前公布的方法进行比较。人类浆细胞很难 由于缺乏来自人基质和骨髓的外源性存活因子， 细胞该提议的目的2将使用人源化（NSG-huCD 34）小鼠模型来更好地预测人源化（NSG-huCD 34）小鼠的潜在免疫应答。 基因编辑的浆细胞疗法的移植潜力。这种人源化小鼠模型也可作为 人嗜性EBV感染的模型。我建议验证抗EBV分泌浆细胞的功能 通过显示这些细胞保护人源化小鼠免受高剂量静脉内EBV攻击。这将 这是第一次显示基因编辑的原代人类浆细胞可以保护人类免受热带疾病的侵害。 在人源化小鼠模型中研究了这种疾病，并将作为使用基因编辑血浆的概念证明。 细胞以提供mAb。 这个项目是理想的，我的训练作为一个年轻的医生科学家，由于我强烈的兴趣，翻译 免疫学，补充我以前在嵌合抗原受体修饰的T细胞 研究，大卫罗林斯博士和理查德詹姆斯博士在B细胞生物学和 导师，以及华盛顿大学的高质量研究和医学教育。的活动 详细介绍了这一建议将提供一个强大的背景，为未来的职业生涯作为一个医生科学家追求 转化免疫学研究。

ABSTRACT Monoclonal antibody (mAbs) therapies have proven to be effective treatments for a multitude of clinical applications. Several of these treatments require repeated administration through intravenous infusions leading to high costs and poor patient adherence. A new field has emerged that seeks to generate mAb-secreting plasma cell therapeutics that could be administered once to provide lifelong therapeutic mAb levels as a therapeutic alternative. Plasma cells have high secretory capacities, can last a lifetime and are thought to be relatively quiescent. Advances in gene editing and ex vivo B cell differentiation have enabled the ability to generate plasma cells that secrete an exogenous mAb. However, current gene editing strategies do not fully account for endogenous antibody expression or control for what isotype the exogenous mAb is expressed as. Additionally, the field of gene-edited human plasma cell lacks both large and small animal models to test the engraftment and functionality of these mAb-secreting plasma cells. Aim 1 of this proposal will use a new gene engineering strategy to generate a human plasma cell therapeutic that secretes an engineered IgG mAb against Epstein Barr Virus (EBV) as a proof of concept for future mAb- secreting cell therapies. I will characterize the ability of gene-edited primary plasma cells to produce large quantities of defined mAbs and compare to previously published methods. Human plasma cells are difficult to engraft in small animal models due to the lack of extrinsic survival factors from human stromal and myeloid cells. Aim 2 of this proposal will use a humanized (NSG-huCD34) mouse model to better predict the potential engraftment potential of gene-edited plasma cells therapies. This humanized mouse model also serves as a model for human-tropic EBV infection. I propose to validate the functionality of anti-EBV secreting plasma cells by showing that these cells protect humanized mice against high dose intravenous EBV challenge. This would be the first time that gene-edited primary human plasma cells were shown to protect against a human-tropic disease in a humanized mouse model and would serve as a proof of concept for use of gene-edited plasma cells to provide mAbs. This project is ideal for my training as a young physician scientist due to my strong interest in translational immunology, the complementation of my previous training in chimeric antigen receptor- modified T cell research, the extensive background of Dr. David Rawlings and Dr. Richard James in B cell biology and mentorship, and the quality research and medical education at the University of Washington. The activities detailed in this proposal will provide a strong background for a future career as a physician scientist pursuing translational immunology research.