A new strategy to eliminate HIV-1-infected cells by unlocking the Env trimer

项目摘要 消除HIV-1感染细胞的困难是根除战略的主要障碍。这是由于 几个因素，包括封闭脆弱的HIV-1包膜糖蛋白（Env）表位， 可以被能够有效的Fc效应子功能的抗体（Ab）识别，例如抗体依赖性细胞免疫应答。 细胞毒性（ADCC）。通过这种应用，我们提出工程化特异于高度保守的CD 4诱导的抗体。 （CD 4 i）Env表位来克服这些障碍并促进通过ADCC消除HIV-1感染的细胞。 因此，我们的中心假设是，CD 4 i治疗性Ab（tAb）将能够更有效地治疗肿瘤。 在HIV+血浆存在的情况下，消除感染的原代CD 4+细胞的能力比同类产品中最好的广泛 中和抗体（bnAb），并因此可以发展成新的tAb家族，其将有效减少 并最终消除HIV-1（PLWH）感染者体内的病毒储存库。拟议的研究建立 在我们成功地从弱中和或CD 4 i Ab开发新的Ab-CD 4杂交体后， 辅助受体结合位点（CoRBS）或高度保守的非中和gp 120内结构域簇A区。我们 前导CoRBS和A簇Ab-CD 4杂交体有效地消除了感染原代HIV-1分离株的细胞， 体外和离体，证明它们可以规避受感染细胞和病毒体上表位的封闭。 此外，它们还能够利用HIV+血浆中存在的Ab的ADCC活性，进一步增强HIV+抗体的抗HIV活性。 ADCC。最后，将抗簇A Ab-CD 4杂合体（例如，A32-CD 4）也抑制了病毒在hu小鼠中的反弹， 以Fc依赖性方式显著减少整合的HIV DNA。在拟议项目中，我们将 工程化具有其他CD 4 i特异性的Ab-CD 4，并且还用有效的小分子CD 4 模拟化合物（CD 4 mc）。这些下一代Ab-CD 4 mc缀合物将有效地敏化HIV-1 感染细胞对ADCC介导的杀伤，提供了一种新的工具，以减少HIV-1感染的细胞库， PLWH。目的1设计制备具有其它CD 4 i特异性的Ab-CD 4杂合体，包括簇A、C11-C12、C11-C14和C11-C14。 gp 41主要免疫优势区。目的2将开发Ab-CD 4 mc缀合物，其中 Ab-CD 4的CD 4部分将被新一代的小化合物CD 4 mcs取代，包括BNM-III-CD 4 mcs。 170和/或更多的活性CD 4 mcs。CD 4 mc将通过PEG接头使用新的连接剂连接至Ab。 tRNA抑制系统。将在体外和离体评价Ab-CD 4/CD 4 mcs中和 主要病毒和消除HIV-1感染的细胞单独或协同与HIV+血浆，在体外和 离体。Aim 3将测试最佳工程变异体减少病毒储存库大小的能力， 移植有来自PLWH的PBMC的人源化小鼠。这一最终目标将评估最有前途的国家的能力， 工程化变体以消除体内感染的细胞并延迟病毒反弹。总的来说，该项目将产生 一种新的强效tAb家族，能够消除HIV-1感染细胞，为不断增长的武器库增添新武器。 旨在实现HIV-1的功能性治愈。

Project Abstract The difficulty in eliminating HIV-1-infected cells is a major roadblock in eradication strategies. This is due to several factors which includes the occlusion of vulnerable HIV-1 envelope glycoprotein (Env) epitopes that can be recognized by antibodies (Abs) capable of potent Fc-effector functions such as Antibody-Dependent Cellular Cytotoxicity (ADCC). With this application we propose to engineer Abs specific for highly conserved CD4 induced (CD4i) Env epitopes to overcome these obstacles and facilitate the elimination of HIV-1 infected cells via ADCC. Accordingly, our central hypothesis is that CD4i therapeutic Abs (tAbs) will be capable of more effective elimination of infected primary CD4+ cells in the presence of HIV+ plasma than the best in class broadly neutralizing Abs (bnAbs) and can thus be developed into a new family of tAb that will be effective in decreasing and ultimately eliminating the viral reservoir in people living with HIV-1 (PLWH). The proposed research builds upon our successful development of new Ab-CD4 hybrids from weakly neutralizing or CD4i Abs specific for the coreceptor binding site (CoRBS) or highly conserved, non-neutralizing gp120 inner domain Cluster A region. Our lead CoRBS and Cluster A Ab-CD4 hybrids efficiently eliminate cells infected with primary HIV-1 isolates both in vitro and ex vivo, demonstrating that they can circumvent the occlusion of epitopes on infected cells and virions. Furthermore, they are also able to harness the ADCC activity of Abs present in HIV+ plasma, further enhancing ADCC. Finally, an anti-Cluster A Ab-CD4 hybrid (e.g., A32-CD4) also inhibited viral rebound in hu-mice and led to a significant reduction in integrated HIV DNA in an Fc-dependent manner. In the proposed project, we will engineer Ab-CD4s of other CD4i specificities and also replace the CD4 moiety with potent small molecule CD4 mimetic compounds (CD4mc). These next generation Ab-CD4mc conjugates will efficiently sensitize HIV-1 infected cells to ADCC-mediated killing, providing a new tool to decrease the HIV-1-infected cell reservoir in PLWH. Aim 1 is designed to prepare Ab-CD4 hybrids of other CD4i specificities including the Cluster A, C11- like and the gp41 Principal Immunodominant regions. Aim 2 will develop Ab-CD4mc conjugates in which the CD4 moiety of the Ab-CD4 will be replaced by a new generation of small compound CD4mcs including BNM-III- 170 and/or more active CD4mcs. The CD4mc will be attached to the Ab through a PEG linker using a novel tRNA suppressor system. Ab-CD4/CD4mcs will be evaluated in vitro and ex-vivo for their ability to neutralize primary viruses and eliminate HIV-1-infected cells alone or synergistically with HIV+ plasma, both in vitro and ex-vivo. Aim 3 will test the capacity of the best engineered variants to reduce the size of viral reservoir in humanized mice engrafted with PBMC from PLWH. This final aim will evaluate the capacity of the most promising engineered variants to eliminate infected cells in vivo and delay viral rebound. Overall, the project will generate a new family of potent tAbs able to eliminate HIV-1 infected cells to add new weapons to the growing arsenal aimed at achieving a functional cure of HIV-1.