Bi-functional Therapeutics for Allergy-BFTA

描述(申请人提供)：免疫球蛋白介导的过敏性疾病，折磨25%的美国人口，表现为过敏性哮喘、鼻炎、食物过敏、特应性皮炎和过敏反应。除了各种经典药物的对症治疗外，针对IgE介导的免疫反应的治疗主要有两种方式：变应原脱敏或特异性免疫治疗(SIT)和抗IgE被动抗体治疗或Xolair。在此，我们基于最近的实验室观察提出了一种泛IgE疗法(PIT)，因为跨越GFP桶的人IgE的受体结合CH(2和CH(3)连接环(C(2-3L))是构象受限的。此外，针对C(2-3L)的抗体限制在GFP(-桶)内，抑制IgE与Fc(RI)的结合(并阻止肥大细胞激活。这一观察结果提示了使用C2-3L-GFP作为泛IgE疗法(PIT)治疗IgE介导的过敏性疾病的产品概念。 C(2-3L-GFP)的双功能免疫原性是激活C(2-3L)中和抗体和辅助性T细胞与GFP支架结合的先决条件。这一前提条件是药物PIT的有效性和安全性的基础，因为：(I)由IgE和抗C(2-3L)形成的免疫复合物与Fc(RI)(肥大细胞/嗜碱性粒细胞上的受体)安全地隔离，并从循环和粘膜隔室中移除。(Ii)由于水母与哺乳动物的进化距离较远，GFP的免疫原性提供了必要的辅助作用，而不会引发对哺乳动物重要宿主蛋白的抗体反应。研究的总体目标是评估PIT的质量单位(纯度)及其生物等效性(生物反应性；强度/浓度)、其安全性(不良反应，ADE)、药代动力学(分布)和药效学(有效性/有效性)作为药物物质。来自适当的TG动物模型的数据将被收集、分析，并打算作为临床替代物。 目的1.评价PIT的化学质量单位和生物等效性。在PIT药材的一致性、安全性、效力和有效性的质量单位表征的所有阶段都需要增量质量。GFP具有显著的热稳定性(差示扫描量热法的Tm为82.6℃)。重组蛋白将通过内部组氨酸标签进行浓缩。聚集性或变性蛋白质可用S-200HR色谱柱和Superdex 75柱层析分离，然后根据电荷密度的不同进行Mono-Q分离，并根据等电点的不同进行层析聚焦。然后通过氨基酸测序、CD、MS/MS和核磁共振对药物材料进行分析。该质量单位将在小鼠身上进行测试，并在另一个物种--大鼠和/或猴子身上进行升级。 目的2.评价PIT的安全性和治疗注意事项 将评估药物物质PIT与天然GFP的三个安全标准：适合保护窗口的抗体反应持续时间、过敏性、自身创造力。我们将评估最后一次通过粘膜途径与全身途径接种PIT方案后抗C(2-3L)抗体应答的持续时间是否在一到三个月的保护窗口内。如果抗体反应在PIT终止后持续六个月以上，我们将重新评估剂量和加强免疫计划。将测定循环免疫复合体、抗GFP IgE和自身免疫抗体的水平。 目的3.PIT法评价免疫球蛋白清除在动物模型中的药代动力学 免疫球蛋白E受体参与免疫球蛋白E的隔离和/或周转。表达人Fc(RI(TG)的小鼠模型用于研究被动转移的人IgE的清除，在最后一次PIT方案免疫后第7天，经静脉或经鼻内途径，通过切断或粘膜途径给药。将评估血清和BAL液中游离JW8和抗IgE/JW8免疫复合体的水平。采用亚类特异性酶联免疫吸附试验检测循环免疫球蛋白和粘膜免疫球蛋白A抗IgE抗体水平。保护性抗-C(2-3L)的持续时间将通过连续两个月给药新鲜的人IgE来评估。 AIM4.PIT在转基因细胞和人肥大细胞体内的药效学研究 来自不同物种的抗血清或亲和纯抗C(2-3L)对人FC(RI(+))的保护作用将在体外测试来自TG小鼠的骨髓来源的肥大细胞和人的肥大细胞。此外，还将利用Fc(RI)(TG小鼠)研究PIT在减轻IgE介导的与气道高反应性(AHR)有关的炎症反应、通过改变细胞外基质(ECM)和收缩问题而导致的气道重塑以及炎症细胞浸润和细胞因子方面的药效学作用。总之，通过靶向IgE恒定区，PIT方案可能会减轻由多种变应原引起的IgE介导性疾病。作为单一的生物制剂，PIT可能包括多种重组变应原的治疗适应症，以满足过敏患者的亚群需求。从这项研究收集的数据可用于准备IND的一部分，以供CBER相关办公室审查。 公共卫生相关性：双功能泛IgE疗法项目可能产生一种可商业化的产品，用于治疗不同适应症的IgE介导的变态反应性疾病。

DESCRIPTION (provided by applicant): IgE-mediated allergic diseases, afflicting 25% of the US population, are manifested as allergic asthma, rhinitis, food allergy, atopic dermatitis, and anaphylaxis. In addition to symptomatic treatments with various classical pharmaceutical agents, there are two main modalities of therapeutic treatment targeting directly at IgE- mediated immune response: Allergen desensitization or specific immunotherapy (SIT) and anti-IgE passive antibody treatment or Xolair. Herein, we propose a pan-IgE therapeutics (PIT) based on recent laboratory observation in that the receptor-binding CH(2 and CH(3 linker loop (C(2-3L) of human IgE, spanning the (-barrel of GFP is, conformationally constrained. Further, antibodies directed to C(2-3L constrained within the (-barrel of GFP, inhibit IgE binding to Fc(RI( and prevent mast cell activation. This observation prompts the product concept of employing C2-3L-GFP as a pan-IgE therapeutics (PIT) in treating IgE-mediated allergic diseases. Bifunctional immunogenicity of C(2-3L-GFP is a prerequisite for activating both neutralizing antibodies to C(2-3L and helper T-cells to GFP scaffold. This prerequisite underlies effectiveness as well as safety of the drug substance PIT in that, (i) Immune complexes formed by IgE and anti-C(2-3L are safely sequestered from binding to Fc(RI( receptors on mast cells/basophils, and are removed from the circulation and mucosal compartments. (ii) Due to the evolutionary distance of jellyfish from the mammals, immunogenicity of GFP provides necessary helper effect without provoking antibodies reactive to vital mammalian host proteins. The overall goal of Research is to evaluate the mass unit (purity) and its biological equivalents of PIT (bioreactivities; strength/concentration), its safety (adverse effect, ADE), pharmacokinetics (distribution), and pharmacodynamics (efficacies/effectiveness) as the drug substance. Data from an appropriate tg animal model will be collected, analyzed, intended as a clinical surrogate. AIM 1. TO EVALUATE THE CHEMICAL MASS UNIT OF PIT AND BIOLOGICAL EQUIVALENCE Incremental quality is required throughout all phases of mass unit characterization for consistency, safety, potency and effectiveness of the drug substance of PIT. GFP exhibits salient thermostability (Tm at 82.6¿C by differential scanning calorimetry). Recombinant protein will be enriched via internal His-tag. Aggregated or denatured protein will be separated by sizing with S-200 HR column and Superdex 75, followed by mono-Q on difference of charge density and by chromatofocussing according to difference of isoelectric point. The drug material will then be analyzed by amino acid sequencing, CD, MS/MS, and NMR. The mass unit will be tested in mice and escaladed in another species, rats and/or monkey. AIM 2. TO EVALUATE THE SAFETY AND THERAPEUTIC PRECAUTION OF PIT Three safety standards of the drug substance PIT vs. the native GFP will be evaluated: duration of antibody response appropriate for the protective window, allergenicity, autocreativity. We will evaluate whether duration of anti-C(2-3L antibody responses following the last immunization of the PIT regimen via mucosal vs. systemic route may be within one to three months' protective window. Should antibody responses persist beyond six months after termination of PIT, we will re-evaluate dosages and the booster schedules. Levels of circulation immune complexes, anti-GFP IgE and autoimmune antibodies will be measured. AIM 3. TO EVALUATE PHARMACOKINETICS OF IGE CLEARANCE IN ANIMAL MODEL BY PIT IgE receptors contribute to sequestration and/ or turnover of IgE. A mouse model expressing human Fc(RI( tg is employed for studying clearance of passively transferred human IgE, administered intravenously or via the intranasal route in a bolus on day 7 after the last immunization of the PIT regimen via subcut vs. Mucosal route. Levels of free JW8 vs. anti-IgE/JW8 immune complexes in sera and the BAL fluid will be evaluated. Levels of circulating IgG and mucosal IgA anti-IgE antibodies will be measured by subclass-specific ELISA. Duration of protective anti-C(2-3L for further rounds of IgE clearance will be assessed by two consecutive monthly administrations of fresh human IgE. AIM4. TO EVALUATE THE PHARMACODYNAMICS OF PIT IN TRANSGENIC AND HUMAN MAST CELLS IN VITRO AND IN VIVO Protection of antisera or affinity pure anti-C(2-3L from different species will be tested on human Fc(RI(+ bone marrow-derived mast cells from tg mice, and human mast cells in vitro. Moreover, Fc(RI( tg mice will be employed for studying pharmacodynamic effect of PIT in attenuating the IgE-mediated inflammatory responses with regard to airway hyperreactivity (AHR), airway remodeling via changes of extracellular matrix (ECM) and contractile issues, and inflammatory cell infiltrate and cytokines. In conclusion, by targeting IgE constant regions, PIT regimens may alleviate IgE- mediated diseases by a myriad of allergens. It is possible that PIT as a single biologics may include therapeutic indications of numerous recombinant allergens catering for subsets of allergic patients. Data collected from this study may be employed in part of preparing IND to pertinent Office for review at CBER. PUBLIC HEALTH RELEVANCE: The Project of bi-functional pan-IgE therapeutics may yield a commercializable product for treating IgE-mediated allergic diseases of different indications.