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dc.contributor.advisorGoldstein, Harris
dc.contributor.authorBardhi, Ariola
dc.identifier.citationSource: Dissertations Abstracts International, Volume: 80-02, Section: B.;Publisher info.: Dissertation/Thesis.;Advisors: Goldstein, Harris.en_US
dc.description.abstractThe development of a functional cure against HIV-1 infection is prevented by the ability of the virus to evade immune responses by mutating rapidly and establishing latency. Treatment of HIV-1-infected patients with latency reactivation agents did not reduce the size of the latent reservoir, indicating that the endogenous patient HIV-1- specific immune responses alone were insufficient to eliminate the reactivated latent HIV-1-infected cells. Consequently, a functional cure for HIV-1 infection would require the development of novel therapies to facilitate the elimination of reactivated latent infected cells. HIV-1-specific antibodies emerge several weeks after acute HIV-1 infection and evolve continuously as a result of a constant virus-antibody race, during which HIV-1 mutates to escape antibody recognition and antibodies undergo new rounds of affinity maturation to target the new mutants in response. While virus neutralization is an important mechanism for HIV-1 control, antibodies can also bind to HIV-1 envelope protein expressed by infected cells and mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate HIV-1-infected cells, and thereby delay disease progression. Highly effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacity of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, our collaborators constructed LSEVh-LS-F, a bispecific multivalent antibody-based fusion protein targeting the HIV-1 envelope receptor and co-receptor binding areas with enhanced ADCC capacity resulting from the defocusylation of the Fc domain. We demonstrated that LSEVh-LS-F has potent and broad neutralizing activity in human peripheral blood mononuclear cells (PBMC) against a range of HIV-1 isolates including envelopes that are resistant to previously described broadly neutralizing antibodies (bNAbs), VRCOI and 3BNC117. This potent activity was confirmed in vivo in humanized mouse and macaque models. We further developed a novel humanized mouse model to evaluate NK cell-mediated ADCC. This model was utilized to demonstrate that LSEVh-LS-F rapidly mobilizes human NK cells to eliminate 80% of HIV-1-infected cells in vivo day after its administration. Despite the short in vivo half-life of the antibody, the potent activity of LSEVh-LS-F supports its potential use as a novel immunotherapeutic agent to eliminate reactivated latent cells. In addition to ADCC, alternative immune approaches should facilitate the elimination of HIV-1 infected cells. One potential approach is utilizing Chimeric Antigen Receptor (CAR) T cells that are engineered to target HIV-1-infected cells. CAR T cells are generated by engineering T cells to express an antibody domain recognizing HIV-1 envelope proteins connected to a CD3zeta signaling domain, which activates specific killing of the infected cell upon antibody binding. Our collaborators used this approach to develop monospecific, bispecific, and trispecific CAR constructs using the two domains from LSEVh-LS-F that bind to the HIV-1 gp120 receptor and co-receptor binding regions, as well as a T20-derivative fusion inhibitor. Additionally, CAR constructs with two different gp120 binding and signaling approaches were developed: mono CARs, in which all the HIV-1 binders are expressed as multifunctional linked proteins on the cell surface and signal together, and duo CARs, where different gp120 binders are grouped into two different surface proteins that signal through two different zeta and/or 4-1BB domains. We performed in vitro studies in human PBMCs, which demonstrated that all gp120-specific CAR constructs protect engineered T cells from HIV-1 infection and have potent activity against HIV-1 infected cells. Furthermore, our data demonstrated that duo CAR T cells have the highest capacity to eliminate cells infected with 11 different HIV-1 strains isolated from different worldwide geographic regions. Finally, studies in humanized NSG mice co-injected with HIV-1 and human PBMC intrasplenically, showed that intrasplenic treatment with bispecific and trispecific CAR T cells reduced HIV-1 infection by 99% in vivo. Overall our work has contributed towards the development of novel therapies to control and reduce HIV-1-infected cells. We have shown that LSEVh-LS-F, a bispecific antibody targeting highly conserved HIV-1 envelope epitopes with enhanced ADCC capacity, has broad and potent HIV-1 activity in vivo . We further used the two antigen-recognition domains from LSEVh-LS-F to design different CAR T cells for a more efficient elimination of HIV-1 infected cells. Our data demonstrates that duo CARs eliminate 99% of HIV-1 infected cells when tested against 11 different viruses spanning different clades. Our in vivo studies further support these data, as we report 99% reduction in HIV-1 infection levels after treatment with bispecific and trispecific duo CAR T cells. The design of trispecific constructs will likely contribute to the generation of effector cells that overcome the limitation of viral escape mutants and therefore the development of more efficient anti-HIV-1 therapies.en_US
dc.publisherProQuest Dissertations & Theses Globalen_US
dc.titleHacking the Immune System to Cure HIV-1: Potent Elimination of HIV-1-Infected Cells by Antibody Based Therapeuticsen_US

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