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

该提议检验了高亲和力IgE的持续的、有限的体内交联 肥大细胞（MC）和嗜碱性粒细胞（BA）上的FcεRI受体可迅速使这些细胞脱敏， 有效预防IgE介导的疾病，如食物过敏和过敏反应。快速脱敏 传统上是一种程序，其中来自IgE介导的对特定 通过暴露于增加过敏原浓度，使过敏原暂时对过敏原无反应 超过几个小时。我们通过增加抗FcεRI α的浓度， 链mAb。这些研究表明：（1）抗Fc εRIα单克隆抗体RD比抗Fc εRIα单克隆抗体RD更有效、更安全、更持久， RD与过敏原;（2）RD与抗Fc εRIα mAb是抗原非特异性的;（3）MC无反应性可以安全地 通过维持该mAb的血清水平来维持;（4）该过程适用于抗人（hu）FcεRIα mAb 在表达hu的小鼠中，而不是小鼠FcεRIα（huFcεRIα小鼠）;和（5）RD依赖于诱导 MC无能和MC FcεRI和IgE的清除。最近，我们发现IgE介导的过敏反应可以是 在huFcεRIα小鼠中，通过重复注射<1 µg IE7（一种小鼠mAb），完全抑制，无不良反应 与huFcεRIα结合，而不管FcεRI与IgE的结合。这些小鼠也可以安全和剂量- 通过单次注射单价（mv）形式的IE7依赖性脱敏，所述IE7具有人IgG 1衍生的 恒定区（mv huIgG 1 IE7）。mv huIgG 1 IE7也消耗血液BA，并去除约90%的MC IgE，但 FcεRIα含量低。MC IgE的丢失，特别是与FcεRI相比不成比例的丢失， 这是出乎意料的，因为IE7不会导致MC在体外失去IgE，并且如果 Ab通过聚集或通过与细胞Fcγ或补体受体结合而变成多价。 该提案建立在这些观察结果的基础上。目的1采用流式细胞术、结构研究、抗Fc γR单克隆抗体， 和mv IE7的hu IgG同种型转换变体，以确定mv huIgG 1 IE7是否在体内交联FcεRI 如果是，通过何种机制。目的1还评估了mv huIgG 1 IE7聚集或 负载IgE的FcεRI的内化是MC IgE在体内不成比例损失所必需的。目标2适用 目的1结果使用mv IE7安全地脱敏主动致敏、超过敏的huFcεRIα小鼠， 突变的IL-4受体以及被动致敏的hu脐带血重建的，hu分泌的， 免疫缺陷小鼠产生大量活化的hu MC和BA;这两种模型都被 难以随着二价IE7剂量的增加而脱敏。AIM 3应用AIM 2结果对恒河猴进行脱敏 猴，其表达与IE7反应的FcεRIα。总之，这些目标应该优化使用mv抗- FcεRIα mAb用于RD的研究，并提示其是否以及如何用于抑制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.