Thwarting HIV evasion of antibody avidity with novel antibody architectures

利用新型抗体结构阻止 HIV 逃避抗体亲合力

• 批准号: 8702139
• 负责人: Pamela J Bjorkman
• 金额: $ 80.19万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702139
• 关键词: AIDS/HIV problem; Affinity; Affinity Chromatography; Antibodies; Antibody Avidity; Antibody Formation; Architecture; Avidity; Binding; Binding Proteins; Binding Sites; Coupled; Cross-Linking Reagents; DNA; Geometry; HIV; HIV Infections; Highly Active Antiretroviral Therapy; Human; Immunity; Lateral; Length; Libraries; Link; Linker DNA; Mediating; Methodology; Mutation; Passive Immunization; Pharmaceutical Preparations; Proteins; Public Health; Reagent; Site; Testing; Vaccines; Virus; abstracting; base; crosslink; density; design; ds-DNA; gene therapy; monomer; neutralizing antibody; novel; prevent

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 density irrelevant. Analogous to using ~3 drugs during HAART therapy, simultaneous binding to several different sites/monomer would reduce/abrogate sensitivity to spike mutations.

描述 抽象的： 尽管努力数十年，但目前尚无疫苗在阻断艾滋病毒感染的浓度下中和抗体。除了促进抗体进化的HIV包膜尖峰的结构特征外，我们还建议HIV尖峰的低密度和有限的横向迁移率阻碍了抗体的二价结合。产生的主要单价结合可最大程度地减少亲和力结合和潜在的神经化，从而扩大了允许抗体进化的HIV突变范围。 HIV尖峰三聚体几何形状不允许通过天然二价抗体进行尖峰交联，但是我们可以构建能够与基因治疗和/或被动免疫抑制的尖峰结合具有高避免性三价结合的蛋白质。我们将设计，表达和测试三聚体尖峰交联试剂，这些试剂与每个尖峰单体（每个三聚体6-9个位点）结合到两个或三个非重叠位点。选择HIV结合蛋白以及如何将其链接起来，将从15-30 HIV尖峰结合蛋白的库开始，结合了双链DNA识别标签。这些将使用可变长度DNA链接，形成双特异性试剂，这些试剂被不同的距离隔开。组合的双特异性试剂将通过亲和力色谱分离，以分离最紧的结合对，将对PCR放大/测序，以确定两个蛋白质成分和接头长度。在识别接头的最佳长度后，将DNA替换为可比的蛋白接头，而两结合蛋白的试剂将与HIV结合文库的第三个结合蛋白相关联。首先通过DNA接头连接三聚化基序以确定最佳长度，然后使用基于蛋白质的接头，从而使最佳的两成分HIV结合蛋白将进行修剪。三聚试剂的内部尖峰 交联将降低灭菌所需的浓度，使艾滋病毒的低 尖峰密度无关。类似于在HAART治疗期间使用〜3种药物的类似 与几个不同位点/单体的结合将降低/消除对峰值突变的敏感性。