Conformational Stabilization of the HIV-1 Env Trimer

描述（由申请方提供）：Avatar正在开发基于可溶性天然Env gp 140三聚体的双酪氨酸（DT）交联的HIV疫苗免疫原。几种最有效的广泛中和Ab（“bnAb”）优先结合天然Env三聚体，因此三聚体免疫原将呈现广泛的关键中和表位，并同时使免疫应答远离免疫显性的非中和表位。然而，开发可溶性三聚体免疫原仍然受到Env三聚体复合物的不稳定性的阻碍。Avatar的DT-三聚体稳定在尖峰的顶点，从而能够完全切割，同时锁定关键的亚稳态中和表位在其中和构象中，从而集中和优化Ab应答。 在我们的I期研究中，我们将靶向DT交联引入gp 140三聚体;并证明我们的DT-Env三聚体保留了天然抗原性。我们的II期研究将通过将DT交联转移到来自多个进化枝A、B和C菌株的Env中并测试影响构象异质性的突变来进一步优化DT-Env三聚体免疫原的设计。这些研究以及伴随的表达、纯化和交联过程的优化将能够选择3-4种优化的候选免疫原用于III期临床前试验。 该项目分为三个特定目的：（i）I期DT-Env三聚体的免疫原性检测;（ii）免疫原优化，包括加标物顶点可变区的菌株和构象优化，随后进行进一步免疫原性检测;以及（iii）生产优化，包括表达、纯化和DT交联反应的优化。 创新：Avatar首次使靶向DT交联变得可行，同时保留了gp 140 Env三聚体的结构和功能完整性，并提高了热稳定性。在蛋白质/复合物在三聚体的顶点处完全折叠后引入靶向交联：免疫原被锁定在抗原上有利的构象中，设计为优化关键保护性表位的展示，同时将免疫应答集中在远离免疫显性的非中和表位。 重要性：紧凑和稳定的DT-gp 140三聚体免疫原的设计将使免疫应答集中于广泛中和的表位，而远离非中和的表位。DT介导的选择Env构象异构体的构象锁定可能进一步具有使“单一构象异构体”免疫原成为可能的潜力。 该项目将由Avatar Medical，LLC和PHRI/UMDNJ的Pinter实验室合作执行。主要目标是在II期期间选择并生产3-4种高质量的候选DT-Env gp 140三聚体免疫原，为临床前开发做准备，并在III期中最终下调最终临床候选物。

DESCRIPTION (provided by applicant): Avatar is developing HIV vaccine immunogens based on dityrosine (DT) crosslinking of soluble, native, Env gp140 trimers. Several of the most potent broadly neutralizing Abs ("bnAbs") preferentially bind native Env trimers, and a trimeric immunogen thus would both present a wide range of key neutralizing epitopes, and simultaneously focus immune responses away from immunodominant, non-neutralizing epitopes. Developing a soluble trimeric immunogen, however, remains hampered by instability of the Env trimer complex. Avatar's DT-trimers are stabilized at the apex of the spike, thereby enabling full cleavage, while locking key, metastable neutralizing epitopes in their neutralizing conformation, thus focusing and optimizing the Ab response. In our Phase I studies, we introduced targeted DT crosslinks into gp140 trimers; and demonstrated that our DT-Env trimers retain native antigenicity. Our Phase II studies will further optimize the design of DT-Env trimer immunogens by transferring DT crosslinking into Envs from multiple Clade A, B and C strains and testing mutations that affect conformational heterogeneity. These studies and accompanying optimization of expression, purification, and crosslinking processes will enable the selection of 3-4 optimized candidate immunogen(s) for preclinical testing in Phase III. The project breaks into three Specific Aims: (i) immunogenicity testing of Phase I DT-Env trimers; (ii) immunogen optimzation, including strain and conformational optimization of the variable regions at the apex of the spike followed by further immunogenicity testing; and (iii) production optimization, including optimization of expression, purification, and the DT crosslinking reaction. Innovations: Avatar for the first time has rendered targeted DT crosslinking feasible, while retaining the structural and functional integrity of the gp140 Env trimer, and also improving thermostability. Targeted crosslinks are introduced after the protein/complex is fully folded at th apex of the trimer: immunogens are locked in antigenically favorable conformations, designed to optimize the display of key protective epitopes, while focusing the immune response away from immunodominant, non-neutralizing epitopes. Significance: the design of compact and stable DT-gp140 trimeric immunogens will focus immune responses toward broadly neutralizing - and away from non-neutralizing - epitopes. DT-mediated conformational locking of select Env conformers may further have the potential to enable 'single conformer' immunogens. The project will be executed in collaboration between Avatar Medical, LLC and the Pinter lab at PHRI/UMDNJ. The main goal is to select and produce 3-4 high-qualify candidate DT-Env gp140 trimeric immunogens during Phase II in preparation for preclinical development, and final down-selection of a final clinical candidate in Phase III.