Conformational Stabilization of the HIV-1 Env Trimer

描述(申请人提供)：阿凡达正在开发HIV疫苗免疫原，基于可溶的、天然的、环境中的gp140三聚体的双赖氨酸(DT)交联。几个最有效的广谱中和抗体(BNAbs)优先与天然环境三聚体结合，因此一个三聚体免疫原既可以提供广泛的关键中和表位，又可以使免疫应答远离免疫优势的非中和表位。然而，由于Env三聚体复合体的不稳定性，开发可溶的三聚体免疫原仍然受到阻碍。阿凡达的DT-三聚体稳定在尖峰的顶端，从而能够完全切割，同时锁定关键的亚稳定中和表位，使其处于中和构象，从而聚焦和优化抗体反应。 在我们的第一阶段研究中，我们在gp140三聚体中引入了靶向DT交联物；并证明了我们的DT-Env三聚体保留了天然的抗原性。我们的第二阶段研究将通过将DT交联物从多个分支A、B和C菌株转移到环境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使选择3-4个优化的候选免疫原(S)用于第三阶段的临床前试验成为可能。 该项目分为三个具体目标：(I)第一阶段DT-Env三聚体的免疫原性测试；(Ii)免疫原优化，包括对尖端可变区的菌株和构象优化，然后进一步进行免疫原性测试；以及(Iii)生产优化，包括表达、纯化和DT交联反应的优化。 创新：阿凡达首次使靶向DT交联成为可能，同时保留了gp140 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/复合体在三聚体的顶端完全折叠后引入靶向交联物：免疫原被锁定在抗原性有利的构象中，旨在优化关键保护性表位的展示，同时将免疫反应从免疫主导的非中和表位集中起来。意义：紧凑和稳定的DT-gp140三聚体免疫原的设计将使免疫反应集中在广泛的中和表位上，而不是非中和表位。DT介导的对选择的Env构象的构象锁定可能进一步有可能使“单一构象”免疫原成为可能。 该项目将由阿凡达医疗有限责任公司和PHRI/UMDNJ的品特实验室合作执行。主要目标是在第二阶段选择和生产3-4个高质量的候选DT-Env gp140三聚体免疫原，为临床前开发做准备，并在第三阶段最终向下选择最终的临床候选。

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.