Structure-based discovery and development of next-generation sCD4 immunotherapeutics for HIV

About This Grant

Abstract Despite major advances in antiretroviral therapy (ART), including long-acting agents such as cabotegravir and lenacapavir, HIV remains incurable due to the persistence of viral reservoirs. Broadly neutralizing antibodies (bnAbs) can mediate post-treatment viral control but are limited by incomplete breadth and viral escape, necessitating complex multi-antibody regimens. Soluble CD4 (sCD4) derivatives represent a mechanistically distinct strategy that exploits HIV’s obligate dependence on CD4 for entry, providing broad neutralization and a high barrier to resistance. In addition, sCD4 engagement exposes CD4-induced (CD4i) epitopes and enables Fc-mediated effector functions to eliminate infected cells. Nevertheless, earlier sCD4 biologics showed limited potency, enhancement of infection of CD4-CCDR5+ cells, and unfavorable pharmacokinetics. We have previously developed a single domain sCD4 (D1 domain) to eliminate MHC-II binding and reduce clearance in vivo. Building on this progress, the overall goal of current project is to engineer a new class of potent, broad, and long-acting sCD4-D1-based bifunctional biologics capable of suppressing HIV replication and promoting clearance of infected cells. We hypothesize that optimizing sCD4-D1 binding kinetics, incorporating CD4i binders, increasing avidity through multi-valency, and engineered IgG1 Fc domains will enhance antiviral potency, breadth, and effector function while mitigating resistance and infection enhancement. To test our hypothesis, we will complete the following aims: 1) Engineer sCD4-D1 variants with enhanced binding kinetics and identify novel single-domain VH binders targeting Env CD4i epitopes using computational structure design, phage display, and time-resolved cryo-EM in an iterative optimization process; 2) Develop and optimize multivalent and bifunctional sCD4-D1-CD4i fusion molecules using structure-guided and generative AI-enabled design approaches, with structural validation by cryo-EM; 3) Evaluate lead candidates for Fc-mediated effector functions and anti- HIV activity in vitro, ex vivo, and in vivo using clinically relevant reservoir clearance assays and humanized NK/T-cell competent mouse models, in the context of both acute infection and ART suppression. We will iterate constructs across aims to reach optimal sCD4-D1-CD4i bispecifics. This work is expected to deliver a transformative class of sCD4-based immunotherapeutics that combine broad neutralization with active elimination of HIV-infected cells. The proposal has the potential to overcome the limitations of ART and bnAbs and advance the field toward durable, ART-free remission. The project aligns with the NIH Office of the Director’s unified strategic priorities in advancing biomedical innovation relevant to public health and HIV/AIDS.