Thwarting HIV evasion of antibody avidity with novel antibody architectures

描述 摘要： 尽管经过几十年的努力，目前还没有疫苗能在阻断HIV感染的浓度下产生中和抗体。除了HIV的包膜刺突的结构特征，有利于抗体逃避，我们建议，低密度和有限的横向流动性的HIV刺突阻碍抗体的二价结合。所得的主要单价结合使亲合力最小化，从而使高亲和力结合和有效中和最小化，从而扩大了允许抗体逃避的HIV突变的范围。HIV刺突三聚体几何结构不允许通过天然存在的二价抗体进行刺突内交联，但我们可以构建能够与刺突高亲合力三价结合的蛋白质，用于基因治疗和/或被动免疫。我们将设计、表达和测试三聚体加标物内交联试剂，其结合每个加标单体的两个或三个非重叠位点（每个三聚体6-9个位点）。选择HIV结合蛋白以及如何连接它们将从15-30个与双链DNA识别标签偶联的HIV刺突结合蛋白的文库开始组合进行。这些将使用可变长度DNA连接以形成由不同距离分开的双特异性试剂。将通过亲和色谱分离合并的双特异性试剂以分离最紧密的结合对，将其进行PCR扩增/测序以确定两种蛋白质组分和接头长度。在鉴定接头的最佳长度后，用相当长度的蛋白质接头替换DNA，并将双结合蛋白试剂连接至来自HIV结合文库的第三结合蛋白。最佳的两组分或三组分HIV结合蛋白将通过首先经由DNA接头连接三聚基序以确定最佳长度，然后使用基于蛋白质的接头来三聚化。三聚体试剂的内钉交联将降低消毒免疫所需的浓度，使艾滋病毒的低钉

DESCRIPTION Abstract: Despite decades of effort, no current vaccine elicits neutralizing antibodies at concentrations blocking HIV infection. In addition to structural features of HIV's envelope spike that facilitate antibody evasion, we propose that the low density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The HIV spike trimer geometry does not allow intra-spike cross-linking by naturally-occurring bivalent antibodies, but we can construct proteins capable of high-avidity trivalent binding to a spike for gene therapy and/or passive immunization. We will design, express, and test trimeric intra-spike cross-linking reagents that bind to two or three non-overlapping sites per spike monomer (6-9 sites per trimer). Choosing HIV-binding proteins and how to link them will be done combinatorially starting with a library of 15-30 HIV spike-binding proteins coupled to double-stranded DNA identifying tags. These will be linked using variable-length DNA to form bispecific reagents separated by different distances. Pooled bispecific reagents will separated by affinity chromatography to isolate the tightest binding pairs, which will be PCR amplified/sequenced to determine the two protein components and the linker length. Upon identifying the optimal length for the linker, the DNA is replaced with a comparable-length protein linker, and the two-binding-protein reagent is linked to a third binding protein from the HIV-binding library. Optimal two- or three-component HIV binding proteins will be trimerized by attaching a trimerization motif first via DNA linkers to determine the optimal length, then using protein-based linkers. The trimeric reagent's intra-spike cross-linking would reduce the concentration required for sterilizing immunity, making HIV's low spike