beta-Amyloid Antibodies with Specific Proteolytic Activity

描述（由申请方提供）：抗淀粉样蛋白β肽抗体[抗Abeta Ab]的被动免疫治疗提供了Abeta肽主动免疫的益处，但没有毒性。该合作项目的目的是开发和测试不仅结合Aa肽而且特异性切割Aa肽的蛋白水解抗Abeta Ab。将使用三种方法来开发蛋白水解性抗Abeta Ab：[1]基于共价反应性Abeta类似物（CRA）的协调非共价识别和不可逆结合，从表达人Ab库的噬菌体文库中选择Ab片段（单链Fv片段），所述共价反应性Abeta类似物（CRA）含有与丝氨酸蛋白酶的活性位点中的亲核试剂反应的化学基团。这种方法已经被验证了其鉴定结合了联合收割机特异性抗原识别与水解肽键能力的Ab的能力。[2]通过以下方式提高抗-A β特异性：（a）随机化重链可变（VH）结构域CDR 3的序列，该区域控制Ab特异性，结合使用CRA的共价亲和力选择;和（B）表达混杂蛋白水解活性的轻链V结构域与来源于特异性抗-Abeta Ab的VH结构域的配对。在每种情况下，将测试scFv克隆体外中和Abeta肽介导的神经毒性的能力。[3]将研究通过用Abeta CRA免疫诱导蛋白水解单克隆抗Abeta Ab，该策略设计用于允许克隆选择产生具有增强的和特异性的抗Abeta蛋白水解活性的Ab的B细胞。Abeta切割位点的测定将鉴定能够切割决定簇（残基25-35）和寡聚肽状态的Ab克隆，所述寡聚肽状态被认为在使用蛋白水解和非蛋白水解Ab的Abeta肽毒性中重要。将在体内测试最具活性的Ab抑制APP/PS1转基因小鼠脑淀粉样蛋白沉积和认知下降的能力。APP/PS1转基因小鼠的RAG-1缺陷系将用于测试人蛋白水解Ab，从而避免混淆异种Ab的鼠免疫应答。能够防止Abeta沉积和认知下降的蛋白水解抗Abeta Ab将是患有AD的人的被动免疫疗法的候选者。

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.