CCR5 immunotoxins as components of HIV cure regimens

该项目将建立CCR5免疫毒素作为HIV宿主耗尽剂的概念验证。 用于治疗策略。艾滋病毒的治疗和缓解战略要求消除或减少艾滋病毒 水库。其他小组寻求的减少储层的方法包括基因编辑，即直接移除 集成的前病毒；延迟反转和伴随的“杀死”策略；以及“阻止和锁定”，这是 通过永久抑制基因表达，有效消除储藏细胞。 相反，我们采取了一种策略，即在感染早期直接清除潜在的存储细胞，假设 大多数早期的水库细胞表达CCR5。使用CD3/CCR5双特异性抗体来耗尽储集层， 我们展示了4/7的延迟反弹和2/7感染SIV的婴儿猕猴的明显治愈。一位治愈了 动物在停用ART后204天进行尸检，第二只动物在1.6年后仍未死亡。两者都有 耗尽了CD8+T细胞以鼓励病毒反弹，但没有看到反弹。没有前病毒DNA 在ART停用后的任何时间点在循环细胞中检测到。敏感的病毒副产物分析失败 恢复复制能力强的病毒。在其中一例处死动物的组织中未检测到前病毒基因组 显然是治愈的动物。因此，到目前为止获得的结果表明，这些动物至少已经实现了 “功能性的”，也许还有灭菌的治疗方法。 我们使用的bsAb可以非常有效地瞬时耗尽CCR5+细胞，但也会导致 炎症反应伴随细胞因子的产生和暂时性的CD3+淋巴细胞减少。因此，滕德尔 开发用于治疗抗艾滋病毒方案的CCR5免疫毒素。我们假设CCR5 免疫毒素可以特异性和低毒地耗尽血液、肠道和其他组织中的CCR5+T细胞 淋巴组织。 SA1.根据CCR5配体-毒素融合产生优化的免疫毒素。以前发布的版本 免疫毒素在体外高浓度下有效，但未能通过体内试验。在这一目标中，我们开发了新的 CCR5配体与不同长度和不同毒素结合的免疫毒素 候选人。然后，我们在体外测试候选细胞对表达CCR5的细胞的特定致死性。 SA2.猕猴发育候选药物的药效学测试，重点是CCR5 在没有免疫激活的情况下从肠道组织中消耗。下面是来自目标1的开发候选者 在猕猴身上进行评估，并将结果与基准bsAb实现的耗竭进行比较，即 已经被证明具有潜在的治愈作用。最重要的是，这些实验测试了候选免疫毒素 在受体动物中，CCR5至少可以在几周内达到近100%的耗竭。 我们的初步数据显示，CCR5+储存库是SIV早期的“阿喀琉斯之踵”，可能是HIV的致命弱点 感染。拟议的研究将对新型Tendel免疫毒素进行概念验证。

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.