Bi-functional Therapeutics for Allergy-BFTA

过敏双功能疗法-BFTA

• 批准号: 7668324
• 负责人: Swey-Shen Chen
• 金额: $ 30万
• 依托单位: IGE THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7668324
• 关键词: A Mouse; Adverse effects; Affinity; Allergens; Allergic; Amino Acid Sequence; Anaphylaxis; Animal Model; Antibodies; Antibody Formation; Antigen-Antibody Complex; Atopic Dermatitis; Attenuated; Autoantibodies; Basophils; Binding; Biological; Blood Circulation; Bolus Infusion; Bone Marrow; CD4 Positive T Lymphocytes; Cell Degranulation; Cells; Charge; Chemicals; Clinical; Complex; Controlled Clinical Trials; Data; Differential Scanning Calorimetry; Disease; Double-Blind Method; Drug Kinetics; Effectiveness; Enrollment; Enzyme-Linked Immunosorbent Assay; Event; Exhibits; Extracellular Matrix; Extrinsic asthma; Food Hypersensitivity; Future; Goals; Green Fluorescent Proteins; Helper-Inducer T-Lymphocyte; Human; Hypersensitivity; IgE; IgE Receptors; Immune Sera; Immune response; Immunization; Immunoglobulin A; Immunoglobulin Constant Region; Immunoglobulin G; Immunotherapy; In Vitro; Inflammatory; Inflammatory Response; Isoelectric Point; Jellyfish; Knowledge; Laboratories; Licensing; Liquid substance; Lung; Mammals; Marketing; Measures; Mediating; Modality; Monkeys; Mono Q; Mucous Membrane; Mus; Pan Genus; Pathogenesis; Patients; Peptide Library; Pharmaceutical Preparations; Pharmacodynamics; Pharmacologic Substance; Phase; Placebo Control; Placebo Effect; Population; Preparation; Protein Sequence Analysis; Proteins; Randomized; Rattus; Recombinant Proteins; Recombinants; Reporting; Research; Rhinitis; Risk; Roentgen Rays; Route; Safety; Scaffolding Protein; Schedule; Serum; Structure; T-Lymphocyte Epitopes; Testing; Therapeutic; Transgenic Organisms; Treatment Protocols; Vaccine Research; Vaccines; Xolair; airway hyperresponsiveness; airway remodeling; anti-IgE; base; cost; cytokine; density; desensitization; dosage; drug development; drug standard; immunogenic; immunogenicity; in vivo; mast cell; mouse model; neutralizing antibody; passive antibodies; prevent; public health relevance; receptor; receptor binding; research study; response; scaffold; success; therapeutic vaccine; thermostability

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.

描述（由申请人提供）：IgE介导的过敏性疾病折磨着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。除了使用各种经典药剂的对症治疗之外，存在直接靶向IgE介导的免疫应答的两种主要治疗性治疗模式：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。本文中，我们基于最近的实验室观察提出了泛IgE治疗剂（PIT），其中跨越GFP的β-桶的人IgE的受体结合CH β 2和CH β 3接头环（C（2-3L）在构象上受到限制。此外，针对限制在GFP的β-桶内的C β 2-3L的抗体抑制IgE与Fc β 1的结合，并防止肥大细胞活化。这一观察提示了采用C2-3L-GFP作为治疗IgE介导的过敏性疾病的泛IgE治疗剂（PIT）的产品概念。 C β 2-3L-GFP的双功能免疫原性是激活针对C β 2-3L的中和抗体和针对GFP支架的辅助T细胞的先决条件。这一先决条件是原料药PIT有效性和安全性的基础，因为（i）由IgE和抗-C β 2-3L形成的免疫复合物被安全地隔离，不与肥大细胞/嗜碱性粒细胞上的Fc β 1受体结合，并从循环和粘膜区室中清除。(ii)由于水母与哺乳动物的进化距离，GFP的免疫原性提供了必要的辅助作用，而不会引起与重要哺乳动物宿主蛋白反应的抗体。研究的总体目标是评价PIT作为原料药的质量单位（纯度）及其生物等效物（生物活性;规格/浓度）、安全性（不良反应，ADE）、药代动力学（分布）和药效学（功效/有效性）。将收集、分析来自适当tg动物模型的数据，预期作为临床替代。 AIM 1.为了验证PIT的化学质量单位和生物等效性，在质量单位表征的所有阶段都需要增量质量，以确保PIT原料药的一致性、安全性、效价和有效性。GFP表现出显著的热稳定性（通过差示扫描量热法，Tm为82.6 ℃）。重组蛋白将通过内部His标签富集。通过S-200 HR色谱柱和Superdex 75进行分级，然后根据电荷密度差异进行mono-Q，并根据等电点差异进行色谱聚焦，从而分离聚集或变性蛋白。然后通过氨基酸测序、CD、MS/MS和NMR分析药物材料。质量单位将在小鼠中进行检测，并在另一种属（大鼠和/或猴）中进行递增。 AIM 2. PIT的安全性和治疗预防 将评价原料药PIT与天然GFP的三个安全性标准：适合保护窗的抗体应答持续时间、过敏性、自体创造性。 我们将评估通过粘膜途径与全身途径的PIT方案的最后一次免疫接种后，抗C β 2-3L抗体应答的持续时间是否在1至3个月的保护窗内。 如果抗体应答持续超过PIT终止后六个月，我们将重新评估剂量和加强方案。 将测量循环免疫复合物、抗GFP IgE和自身免疫抗体的水平。 AIM 3. PIT法测定IGE清除剂在动物模型中的药代动力学 IgE受体有助于IgE的螯合和/或周转。 表达人Fc（RI）β的小鼠模型用于研究被动转移的人IgE的清除，所述人IgE在PIT方案的最后一次免疫接种后第7天静脉内或通过鼻内途径推注给药，所述PIT方案通过亚切途径与粘多糖途径。 将评价血清和BAL液中游离JW 8与抗IgE/JW 8免疫复合物的水平。 将通过亚类特异性ELISA测量循环IgG和粘膜伊加抗IgE抗体的水平。 通过连续两个月给予新鲜人IgE，评估保护性抗-C β 2-3L持续时间，以进行进一步的IgE清除。 AIM4. PIT在转基因细胞和人肥大细胞中的药理作用 来自不同物种的抗血清或亲和纯抗-C β 2-3L的保护作用将在体外对来自tg小鼠的人Fc β 1+骨髓来源的肥大细胞和人肥大细胞进行测试。 此外，将使用Fc（RI）tg小鼠来研究PIT在减弱IgE介导的关于气道高反应性（AHR）、经由细胞外基质（ECM）和收缩问题的变化的气道重塑以及炎性细胞浸润和细胞因子的炎症反应中的药效学作用。 总之，通过靶向IgE恒定区，PIT方案可以减轻由无数过敏原引起的IgE介导的疾病。 PIT作为一种单一的生物制剂可能包括多种重组过敏原的治疗适应症，以满足过敏患者的亚群。 从本研究中收集的数据可用于向相关办公室准备IND，以供CBER审查。 公共卫生关系：双功能泛IgE治疗剂项目可能产生用于治疗不同适应症的IgE介导的过敏性疾病的商业化产品。