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

摘要 单抗(MAbbs)疗法已被证明是治疗多种临床疾病的有效方法 申请。其中几种治疗方法需要通过静脉输液重复给药 高成本和病人依从性差。一个寻求产生分泌单抗的新领域已经出现 浆细胞疗法，可一次性给予，以提供终身治疗性mAb水平作为 另一种治疗方法。浆细胞具有很高的分泌能力，可以持续一生，被认为是 相对平静。基因编辑和体外B细胞分化的进展使人们能够 产生分泌外源性单抗的浆细胞。然而，目前的基因编辑策略并不完全 解释内源性抗体的表达或控制外源性mAb表达为什么同种类型。 此外，基因编辑的人类浆细胞领域缺乏大小动物模型来测试 这些分泌单抗的浆细胞的植入和功能。 这项提案的目标1将使用一种新的基因工程策略来产生一种人类浆细胞疗法 这将分泌一种针对EB病毒(EBV)的工程Ig G mAb，作为未来mAb的概念证明。 分泌细胞疗法。我将描述基因编辑的原代浆细胞产生大量 定义的mAbb的数量，并与以前发布的方法进行比较。人类浆细胞很难 缺乏人基质和髓系细胞外源性生存因子的小动物移植模型 细胞。这项提案的目标2将使用人源化(NSG-huCD34)小鼠模型来更好地预测潜在的 基因编辑的浆细胞疗法的植入潜力。这种人性化的鼠标模型还可以用作 人类嗜人EBV感染模型。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注射EBV的攻击。这将会 这是第一次显示经过基因编辑的原代人类浆细胞可以预防嗜人型病毒 在人源化小鼠模型中的疾病，并将作为使用基因编辑血浆的概念证明 提供单抗的细胞。 这个项目非常适合我作为一名年轻内科科学家的培训，因为我对翻译有浓厚的兴趣 免疫学，对我之前接受的嵌合抗原受体修饰T细胞培训的补充 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。活动 这份提案中详细介绍的内容将为未来的职业生涯提供坚实的背景，成为一名内科科学家 翻译免疫学研究。

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.