We developed novel immunoagonists (synTacs) to selectively activate and expand virus-specific CD8+ T cells in vitro and in vivo. SynTacs consist of dimeric Fc-domain scaffolds linking CD28-specific agonists to HLA-A2 MHC molecules covalently-tethered to a virus-derived peptide enabling delivery of TCR-specific and costimulatory signals to antigen-specific T cells. We hypothesized that synTac-activation of virus-specific effector-memory CD8+ T cells would selectively target them for lentiviral transduction, enabling generation of CAR-T cells with more potent and sustained activity than those produced by standard ?CD3/?CD28 antibody (?CD3/28) activation. Using a duoCAR construct which targets two distinct HIV gp120 epitopes (HIV-CAR) to potently eliminate HIV-infected cells, we determined if selectively engineering HIV-CAR into CMV-specific memory CD8 T cells generated more potent HIV-CAR-T cells than those generated by standard ?CD3/28 polyclonal T cell activation
CD8+ T cells purified from an HIV(-)/CMV(+) donor were activated by ?CD3/28 or CMV-NLV-?CD28-synTac (CMV-synTac), a synTac linked to a CMV-pp65-peptide (NLVPMVATV) and CD28-specific agonist, and transduced with an HIV-CAR lentivector. HIV-CAR-T cells were either cocultured or co-injected into NSG mice with HIV luciferase-infected autologous PBMCs to measure in vitro and in vivo viral suppression
Ten days after CMV-synTac treatment, CMV-specific CD8+ T cells expanded ~70-fold, compared to no expansion after ?CD3/28 treatment. After synTac treatment, the HIV-CAR lentivector selectively transduced CMV-specific CD8+ T cells (96% NLV-tetramer+) as compared to ?CD3/28-activated HIV-CAR CD8+ T cells (8.8% NLV-tetramer+). Perforin expression by synTac-generated HIV-CAR-T cells was 3.2-fold higher than those generated by ?CD3/28. After 2-day coculture, synTac- and ?CD3/28-generated CAR-T cells (E:T ratio of 1:1) suppressed HIV infection by 91.5% and 40.8%, respectively. SynTac-generated HIV-CAR-T cells suppressed HIV infection in humanized mice by 95.9% as compared to untreated animals
CMV-synTac selectively activates and expands CMV-specific CD8 T cells, enabling their selective transduction with an HIV-CAR lentivector and conversion into HIV-CAR-T cells with more potent suppressive HIV activity than standard ?CD3/28-generated HIV-CAR-T cells. CMV-synTac-generated HIV-CAR-T cells potently suppressed HIV infection in humanized mice. These results support a new strategy to generate more potent HIV-CAR-T cells