CCR5 immunotoxins as components of HIV cure regimens

该项目将建立CCR 5免疫毒素作为HIV病毒清除剂的概念验证 用于治疗策略。艾滋病毒治疗和缓解战略需要消除或减少艾滋病毒 水库其他研究小组追求的减少储层的方法包括基因编辑，即，直接去除 整合的前病毒;潜伏期逆转和伴随的“杀死”策略;和“封锁和锁定”，这是 通过永久抑制基因表达有效消除储库细胞。 相反，我们采取了在早期感染中直接消除潜在储库细胞的策略，假设 大多数早期储库细胞表达CCR 5。使用CD 3/CCR 5双特异性抗体进行储库耗竭， 我们证明了4/7只SIV感染的幼年猕猴的延迟反弹和2/7只SIV感染的幼年猕猴的明显治愈。一个固化 在ART停药后204天对动物进行尸检，第二只动物在1.6年后仍保持无病毒血症。两 耗尽CD 8 + T细胞以促进病毒反弹，但未观察到反弹。没有前病毒DNA 在ART停药后的任何时间点在循环细胞中检测到。敏感的病毒生长检测失败 恢复复制能力的病毒在一只被处死的动物的组织中没有检测到前病毒基因组， 显然是治愈的动物因此，迄今为止获得的结果表明，这些动物至少达到了 “功能性”，也许是杀菌治疗。 我们使用的bsAb实现了非常有效的CCR 5+细胞的瞬时消耗，但也引起了细胞凋亡。 炎症反应伴细胞因子产生和暂时性CD 3+淋巴细胞减少。因此，Tendel 开发用于治疗性抗HIV方案的CCR 5免疫毒素。我们假设CCR 5 免疫毒素可以实现来自血液、肠道和胃肠道的CCR 5 + T细胞的特异性和最低毒性的消耗， 淋巴组织 SA 1.基于CCR 5配体-毒素融合产生优化的免疫毒素。此前公布 免疫毒素在体外高浓度下有效，但在体内试验中失败。为此，我们开发了新的 基于CCR 5配体与不同长度接头和各种毒素组合的免疫毒素 候选人然后，我们在体外测试候选物对CCR 5表达细胞的特异性致死性。 SA 2.在恒河猴中测试开发候选药物的药效学，重点是CCR 5 从肠道组织消耗而没有免疫激活。在这里，目标1的开发候选人是 在猕猴中进行评估，并将结果与基准bsAb实现的耗竭进行比较， 已经被证明有潜在的治疗作用。最重要的是，这些实验测试候选免疫毒素 可以在受体动物中实现近100%CCR5消耗至少数周。 我们的初步数据显示，CCR 5+储库是早期SIV和HIV的“阿喀琉斯之踵 感染拟议的研究将寻求新的Tendel免疫毒素的概念验证。

This project will establish proof-of-concept for CCR5 immunotoxins as HIV reservoir-depletion agents for use in cure strategies. HIV cure and remission strategies require elimination or reduction of the HIV reservoir. Approaches to reservoir reduction pursued by other groups include gene editing, i.e., direct removal of integrated provirus; latency reversal and an accompanying “kill” strategy; and “block-and-lock”, which is the effective elimination of reservoir cells by permanently suppressing gene expression. We instead pursued a strategy of directly eliminating potential reservoir cells in early infection, hypothesizing that most early reservoir cells express CCR5. Using a CD3/CCR5 bispecific antibody for reservoir depletion, we demonstrated delayed rebound in 4/7 and apparent cure of 2/7 SIV-infected infant macaques. One cured animal was necropsied 204 days after ART withdrawal and the second remains aviremic after 1.6 years. Both were depleted of CD8+ T cells to encourage viral rebound, but no rebound was seen. No proviral DNA was detected in circulating cells at any time point following ART withdrawal. Sensitive viral outgrowth assays failed to recover replication-competent virus. No proviral genomes were detected in the tissues of one sacrificed and apparently cured animal. Thus, results obtained so far show that these animals have achieved at least “functional” and perhaps sterilizing cure. The bsAb we employed achieves very efficient, transient depletion of CCR5+ cells but also causes an inflammatory reaction with cytokine production and temporary CD3+ lymphopenia. Tendel is therefore developing CCR5 immunotoxins for use in curative anti-HIV regimens. We hypothesize that CCR5 immunotoxins can achieve specific and minimally toxic depletion of CCR5+ T cells from blood, gut, and lymphoid tissues. SA1. Produce optimized immunotoxins based on CCR5 ligand-toxin fusions. Previously published immunotoxins were effective at high concentration in vitro but failed in-vivo tests. In this aim we develop new immunotoxins based on CCR5 ligands combined with linkers of different lengths and with various toxin candidates. We then test the candidates in vitro for specific lethality to CCR5-expressing cells. SA2. Test pharmacodynamics of development candidates in rhesus macaques, with a focus on CCR5 depletion from gut tissues without immune activation. Here the development candidates from Aim 1 are assessed in macaques and the results compared to depletion achieved by the benchmark bsAb, which is already shown to be potentially curative. Most importantly, these experiments test if candidate immunotoxins can achieve nearly 100% CCR5 depletion for at least several weeks in recipient animals. Our preliminary data show that the CCR5+ reservoir is an “Achilles’ heel” in early SIV and perhaps HIV infection. The proposed research will pursue proof-of-concept for novel Tendel immunotoxins.