Rapid, safe suppression of IgE-mediated disease with monovalent anti-ceRIa mAb

这一建议验证了高亲和力IgE在体内持续、有限地交联的假设 肥大细胞(MC)和嗜碱性粒细胞(BA)上的受体FcεRI迅速使这些细胞脱敏，并安全和 有效预防食物过敏、过敏反应等由IgE介导的疾病。快速脱敏(RD) 传统上是一种程序，其中MC和BA来自对特定的IgE介导性过敏的人 过敏原通过暴露在不断增加的过敏原浓度中而暂时对该过敏原失去反应 超过几个小时。我们通过用浓度增加的抗FcεRIα接种小鼠来修改RD 链式单抗。这些研究表明：(1)抗Fc-εRI-α单抗治疗RD有效、安全、持续时间长 (2)抗FcεRIα单抗的RD是非抗原特异性的；(3)MC无反应是安全的 通过维持这种单抗的血清水平来维持；(4)这一过程与抗人(Hu)FcεRIα单抗一起工作 在表达Hu的小鼠中，而不是在小鼠FcεRIα(huFcεRIα小鼠)中；以及(5)RD依赖于 MC无能和MC Fc、εRI和Ig E的清除。最近，我们发现IgE介导的过敏反应可以通过 在huFcεRIα小鼠中，通过重复注射IE7-1微克，完全抑制，没有不良反应。 它与HuFcεRIα结合，而不考虑FcεRI与IgE的关联。这些小鼠也可以安全地和剂量- 一次注射含有人IgG1来源的单价(MV)形式的IE7依赖地脱敏 恒定区(MV HuIgG1IE7)。MV huIgG1 IE7也会耗尽血液基础，并去除~90%的MC IgE，但 小FCεRIα。与FcεRI相比，MC IgE的丢失，特别是其不成比例的损失，是 意想不到的，因为IE7不会导致MCs在体外失去Ig E，并且这种MV抗体应该只有在以下情况下才会使FcεRI交联化 抗体通过聚集或与细胞内Fc、γ或补体受体结合而成为多价抗体。 这项建议是建立在这些观察的基础上的。目的1采用流式细胞术，结构研究，抗FcγR单抗， 用MVIE7和Hu Ig G同型切换变异体确定MVhuIg G1IE7在体内是否与FcεRI发生交联性 如果是，通过什么机制(S)。AIM 1还评估MV huIgG1 IE7聚合或 体内MC免疫球蛋白E的不成比例丢失需要内化携带免疫球蛋白E的FcεRI。目标2适用 目的1应用MV IE7安全脱敏主动致敏的过敏性HuFcεRIα小鼠 突变的IL-4受体以及被动致敏的HU脐带血-重组HU细胞因子分泌， 免疫缺陷小鼠产生大量激活的HU MC和BAs；这两种模型都已被 随着二价IE7剂量的增加，难以脱敏。目的3应用目的2的结果使恒河猴脱敏 猴子，表达与IE7反应的FcεRIα。综上所述，这些目标应该会优化MV的使用 FcεRIα单抗用于RD，并建议是否以及如何使用它来抑制Hu IgE介导的疾病。

This proposal tests the hypothesis that sustained, limited in vivo crosslinking of the high affinity IgE receptor, FcεRI, on mast cells (MCs) and basophils (BAs) rapidly desensitizes these cells and safely and effectively prevents IgE-mediated diseases, such as food allergy and anaphylaxis. Rapid desensitization (RD) is traditionally a procedure in which MCs and BAs from people who have IgE-mediated allergy to a specific allergen are made temporarily unresponsive to that allergen by exposure to increasing allergen concentrations over several hours. We modified RD by inoculating mice with increasing concentrations of an anti FcεRI α chain mAb. These studies showed that: (1) RD with anti-FcεRIα mAb is effective, safer and longer lasting than RD with allergen; (2) RD with anti-FcεRIα mAb is antigen-nonspecific; (3) MC unresponsiveness can be safely sustained by maintaining serum levels of this mAb; (4) this process works with anti-human (hu) FcεRIα mAbs in mice that express hu, rather than mouse FcεRIα (huFcεRIα mice); and (5) RD depends both on induction of MC anergy and removal of MC FcεRI and IgE. Recently, we have found that IgE-mediated anaphylaxis can be fully suppressed in huFcεRIα mice, without adverse effect, by repeatedly injecting <1 µg of IE7, a mouse mAb that binds to huFcεRIα regardless of FcεRI association with IgE. These mice can also be safely and dose- dependently desensitized by a single injection of a monovalent (mv) form of IE7 that has human IgG1-derived constant regions (mv huIgG1 IE7). mv huIgG1 IE7 also depletes blood BAs, and removes ~90% of MC IgE, but little FcεRIα. The loss of MC IgE and particularly, its disproportionate loss compared to FcεRI, was unexpected, because IE7 does not cause MCs to lose IgE in vitro and this mv Ab should only crosslink FcεRI if the Ab becomes multivalent by aggregating or by binding to cellular Fcγ or complement receptors. This proposal builds on these observations. Aim 1 uses flow cytometry, structural studies, anti-FcγR mAb, and hu IgG isotype switch variants of mv IE7 to determine whether mv huIgG1 IE7 crosslinks FcεRI in vivo and, if so, through what mechanism(s). Aim 1 also evaluates whether mv huIgG1 IE7 aggregation or internalization of IgE-loaded FcεRI is required for the disproportionate in vivo loss of MC IgE. Aim 2 applies Aim 1 results to use mv IE7 to safely desensitize actively sensitized, hyperallergic huFcεRIα mice that express a mutant IL-4 receptor as well as passively sensitized hu cord blood-reconstituted, hu cytokine-secreting, immunodeficient mice that develop large numbers of activated hu MCs and BAs; both models have been difficult to desensitize with increasing doses of divalent IE7. Aim 3 applies aim 2 results to desensitize rhesus monkeys, which express FcεRIα that reacts with IE7. Together, these aims should optimize the use of mv anti- FcεRIα mAb for RD and suggest whether and how it could be used to suppress hu IgE-mediated disease.