beta-Amyloid Antibodies w/ Specific Proteolytic Activity

描述（由申请人提供）：抗淀粉样蛋白β肽抗体（anti-Abeta Abs）的被动免疫疗法提供了Abeta肽主动免疫的益处，但没有毒性。合作项目的目的是开发和测试既能结合Aa肽又能特异性切割Aa肽的蛋白水解抗- abeta抗体。三种方法将被用于开发蛋白水解抗Abeta抗体：[1]从噬菌体文库中选择表达人类Ab库的Ab片段（单链Fv片段），基于协调的非共价识别和不可逆结合含有化学基团的共价活性Abeta类似物（CRAs）与丝氨酸蛋白酶活性位点的亲核试剂反应。该方法已被证实能够识别结合特异性抗原识别和肽键水解能力的抗体，通过：(a)随机化重链变量（VH）结构域CDR3（控制Ab特异性的区域）的序列，结合使用CRAs的共价亲和力选择，提高抗abeta特异性；(b)将表达混杂蛋白水解活性的轻链V结构域与来自特异性抗Abeta抗体的VH结构域配对。在每种情况下，scFv克隆将在体外测试其中和Abeta肽介导的神经毒性的能力。通过免疫Abeta CRA诱导蛋白水解单克隆抗Abeta抗体，这一策略旨在允许克隆选择产生具有增强和特异性抗Abeta蛋白水解活性的抗体的B细胞。利用蛋白水解和非蛋白水解抗体，测定Abeta切割位点将鉴定出能够切割决定因子（残基25-35）和寡聚肽状态的Ab克隆，这被认为是Abeta肽毒性的重要因素。在APP/ ps1转基因小鼠中，最活跃的抗体将在体内测试其抑制脑淀粉样蛋白沉积和认知能力下降的能力。APP/ ps1转基因小鼠的rag1缺失系将被用于测试人类蛋白水解抗体，从而避免混淆小鼠对异种抗体的免疫反应。能够阻止Abeta沉积和认知能力下降的蛋白水解抗Abeta抗体将成为人类AD被动免疫治疗的候选药物。

DESCRIPTION (provided by applicant): Passive immunotherapy with anti-amyloid beta peptide antibodies [anti-Abeta Abs] offers the benefits but not the toxicity of active immunization with Abeta peptide. The purpose of collaborative project is to develop and test proteolytic anti-Abeta Abs that not only bind Aa peptide but also cleave specifically the Aa peptide. Three approaches will be used to develop proteolytic anti-Abeta Abs: [1] selection of Ab fragments (single chain Fv fragments) from a phage library expressing the human Ab repertoire based on coordinated noncovalent recognition and irreversible binding of covalently reactive Abeta analogs (CRAs) containing chemical groups reactive with the nucleophiles in the active site of serine proteases. This approach has been validated for its capacity to identify Abs that combine specific antigen recognition with the ability to hydrolyze peptide bonds, [2] Improvement in anti-Abeta specificity by: (a) Randomizing the sequence of the heavy chain variable (VH) domain CDR3, a region governing Ab specificity, combined with covalent affinity selection using the CRAs; and (b) Pairing of light chain V domains expressing promiscuous proteolytic activity with VH domains derived from specific anti-Abeta Abs. In each case, the scFv clones will be tested for their capacity to neutralize Abeta peptide-mediated neurotoxicity in vitro. [3] Induction of proteolytic monoclonal anti-Abeta Abs by immunization with Abeta CRA, a strategy designed to permit clonal selection of B cells producing Abs with enhanced and specific anti-Abeta proteolytic activity will be studied. Determination of the Abeta cleavage site(s) will identify Ab clones capable of cleaving the determinant (residues 25-35) and oligomeric peptide states thought to be important in Abeta peptide toxicity using proteolytic and non-proteolytic Abs. The most active Abs will be tested in vivo for their capacity to inhibit cerebral amyloid deposits and cognitive decline in APP/PS1-transgenic mice. A RAG-1-deficient line of APP/PS1-transgenic mice will be used to test human proteolytic Abs, thus avoiding confounding murine immune responses to xenogeneic Abs. Proteolytic anti-Abeta Abs capable of preventing the deposition of Abeta and cognitive decline would be candidates for passive immunotherapy of humans with AD.