beta-Amyloid Antibodies with Specific Proteolytic Activity

描述(由申请人提供)：用抗淀粉样β多肽抗体进行被动免疫治疗[抗Abeta抗体]提供了Abeta多肽主动免疫的好处，但没有毒性。合作项目的目的是开发和测试既能结合AA多肽又能特异性切割AA多肽的蛋白水解性抗Abeta抗体。将使用三种方法来制备蛋白水解性抗Abeta抗体：[1]从表达人抗体谱系的噬菌体文库中选择抗体片段(单链Fv片段)，基于协同非共价识别和共价反应性Abeta类似物(CRA)的不可逆结合，所述CRA包含与丝氨酸蛋白酶活性部位的亲核试剂反应的化学基团。这种方法已经得到验证，它能够识别结合了特异性抗原识别和水解肽的能力的抗体，[2]通过以下方式提高了抗Abeta的特异性：(A)将控制抗体特异性的重链可变区CDR3的序列随机化，并使用CRA进行共价亲和选择；以及(B)将表达混杂蛋白分解活性的轻链V结构域与来自特定抗Abeta抗体的VH结构域配对。在每种情况下，单链抗体克隆都将在体外测试它们中和Abeta肽介导的神经毒性的能力。[3]通过用Abeta CRA免疫诱导蛋白水解性抗Abeta单抗，旨在允许克隆选择产生具有增强的和特异的抗Abeta蛋白分解活性的Abs的策略将被研究。对Abeta裂解位点的测定(S)将确定能够利用蛋白水解性和非蛋白质水解性抗体切割决定簇(残基25-35)和寡聚肽状态的抗体克隆，这些抗体被认为在Abeta多肽毒性中起重要作用。最活跃的抗体将在体内测试其抑制脑淀粉样蛋白沉积和APP/PS1转基因小鼠认知能力下降的能力。一株RAG-1缺陷的APP/PS1转基因小鼠将被用来测试人类蛋白水解性抗体，从而避免混淆小鼠对异种抗体的免疫反应。蛋白水解性抗Abeta抗体能够阻止Abeta的沉积和认知能力下降，有望成为AD患者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.