PotentIn VivoNK Cell-Mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-Bispecific and Hexavalent Broadly Neutralizing Fusion Protein

Abstract

Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities 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, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate>80% of HIV-1-infected cells 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir. Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

“…In this context, other strategies such as site-directed mutagenesis, bispecific antibody formats and glycan engineering have been explored to try to increase Fc binding to activating receptors and decrease the interaction with the inhibitory receptor FcγRIIb (64)(65)(66)(67). For example, afucosylated antibodies against RSV, Ebola virus and HIV with enhanced FcγR binding showed enhanced efficacy in rodent models (67)(68)(69).…”

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…, 114 , 115 Enhanced binding to the FcγR by these afucosylated antibodies was correlated with enhanced efficacy in murine models 114 115 For example, the afucosylated gp120-bispecific and hexavalent broadly neutralizing fusion protein – LSEVh-LS-F also showed potent inhibition of HIV-1 and simian-HIV infection in humanized mouse and macaque models through NK-cell mediated ADCC 84 …”

“…134 Humanized IgG1   Enhanced tumor inhibition in A549 orthotopic mouse models over WT-huMab-HER3 Palivizumab-NRespiratory syncytial virus (RSV)afucosylatedTransgenic N. benthamiana with RNAi to eliminate the expression of plant specific xylosyl and fucosyl transferase genesEnhanced RSV protection in rat models over fucosylated counterpart(d)Humanized IgG1     h-13F6Heavily glycosylated mucin-like domain of EBOV glycoprotein (GP)afucosylated N. benthamiana plants with RNAi to eliminate the expression of plant specific xylosyl and fucosyl transferase genesEnhanced anti-viral protection over CHO cells-produced fucosylated counterpartHiatt et al, 114 Zeitlin et al, 115 Chimeric IgG1     LSEVh-LS-F Hexavalent fusion protein with IgG1 FcCD4 and HIV-1 gp120-binding sitesafucosylatedGDP-fucose transporter −/− CHO cellsPotent inhibition of SHIV infection in macaque models over PBS controlBardhi et al. 84     Potent in vivo neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117 in humanized mouse KM2760CC chemokine receptor 4 (CCR4)ReducedYB2/0 cellsEnhanced anti-tumor activity over fucosylated counterpart in human PBMC-engrafted mouse modelNiwa et al 135 Chimeric IgG1   Significant anti-tumor activity in disseminated and non-disseminated Sezary syndrome (SS) and Mycosis fungoides (MF) mouse modelsYano et al. 136    <...>…”

Establishment of a Novel Humanized Mouse Model To Investigate In Vivo Activation and Depletion of Patient-Derived HIV Latent Reservoirs

“…This proposed strategy of intrasplenically injecting NSG mice with PBMC obtained from HIV-infected individuals virally suppressed by long-term ART, which we termed Splenic-injected Primary HIV-Infected Reservoir (SPHIR) mice, built upon our previous experience using a humanized mouse model consisting of NSG mice intrasplenically injected with activated human PBMC and HIV to study NK cell-mediated inhibition of acute HIV infection (21,22). We demonstrate here that the SPHIR mouse spleens provide a lymphoid environment wherein latently infected cells from an ART-suppressed donor were rapidly reactivated to produce replication-competent virus which rapidly disseminated infection to human T cells in the spleen which led to viremia within 1 week of intrasplenic injection.…”