Persistence of CMV-specific anti-HIV CAR T cells after adoptive immunotherapy

Abstract

The success of chimeric antigen receptor (CAR)-T cell (Tc) immunotherapy in refractory B-cell acute lymphoblastic leukemia (B-ALL) suggests adaptation of this strategy toward HIV. Because cytomegalovirus (CMV) vaccine vectors generated Tc responses that controlled viral replication, these studies aim to genetically modify CMV-specific Tc with HIV-CAR2 vectors and link HIV immunotherapy to persistent CMV antigen stimulation. To mimic a clinical scenario, rhesus macaques were challenged with the CCR5-tropic simian/human immunodeficiency virus (SHIV-D) prior to antiretroviral therapy (ART). Autologous CMV-specific Tc were transduced with the control CEA-CAR2 or CD4-CAR2/maC46 vectors and reinfused. After stopping ART, the plasma viral load (PVL) in the control rebounded and was sustained above 1.7 × 10⁴ copies/mL; PVL in CD4-CAR2-treated animals was delayed up to 6 weeks and 10-fold lower. The CD4 CAR-Tc frequency peaked at day 7 and was detected in lymphoid tissues at 6 weeks. Both CEA-CAR2 and CD4-CAR2 persisted in PBMCs for about 2 years, which indicates that the CMV-specific CAR Tc were maintained based on their CMV specificity. However, long-term PVL was stable in all animals. Thus, CMV-specific CAR-Tc were active initially, persisted long term, but failed to control viral replication.IMPORTANCEBecause of latent viral reservoirs and a dysfunctional immune response, HIV replication rebounds when antiretroviral therapy is interrupted. Therefore, cytomegalovirus (CMV)-specific Tc were genetically modified with anti-HIV CD4-CAR2 vectors to link the targeting of the HIV envelope to the persistent CMV immune response. In this clinical scenario with simian/human immunodeficiency virus (SHIV) challenge and antiretroviral therapy (ART) suppression, early activity of the CAR Tc delayed rebound in the rhesus macaque/SHIV challenge model. However, even with long-term persistence of CAR Tc in the blood, control of viral replication was not achieved. These data suggest that CAR Tc will require additional interventions to cure HIV infection.

Keywords: CD4-CAR T cells; CMV-specific immune responses; adoptive immunotherapy; persistence and biodistribution; rhesus SHIV-challenge model.

Fig 1

The frequency of rhesus CMV-specific CD8⁺ T lymphocytes in (A) PBMCs ex vivo, (B) bead-stimulated, and (C) CMV peptide-stimulated populations by intracellular cytokine staining. PBMCs were stimulated with CD3/CD28-coated beads (B) or rhesus CMV peptide pools (C) and then expanded for 2 weeks with IL-2. Unstimulated (A) or expanded cells were restimulated with CMV peptides plus anti-CD49d and anti-CD28 costimulatory antibodies overnight in the presence of brefeldin A and then stained for intracellular IFN-γ/TNF-α production. Cells were gated for singlets, live cells, lymphocytes, CD3⁺ cells, and CD8⁺ cells.

Fig 2

Transduction of CMV-specific Tc with retroviral vectors. (A) Schematic diagrams of the vector expressing control CAR (αCEA-CAR2), maC46 (M341), and the αHIV-CAR (CD4-CAR2). (B) Structure of the CD4-CAR2 with the D1-D4 CD4 extracellular (EC) domains, the CD28 transmembrane (TM) domain, and CD28/TCRζ cytoplasmic signaling domains. (C) Clinical-scale expansion and transduction of rhesus CMV-specific T cell populations. Autologous rhesus PBMCs were stimulated with CMV overlapping peptide pools (IE1, IE2, and pp65) and CD28 antibody plus IL-2 (50 IU/mL) and IL-15 (50 ng/mL). On Days 2 and 3, cells were exposed to the retroviral vector coated on retronectin (20 µg/cm²) and expanded for 10 more days. (D) Expression of the CAR vector in CD8⁺ T lymphocytes by flow cytometry.

Fig 3

Plasma viral load before and after CAR2 Tc immunotherapy. To mimic a clinical scenario, rhesus macaques were challenged with the CCR5-tropic chimeric SHIV-D (50 ng SIV Gag p27 per animal) and administered ART (tenofovir 20 mg/kg/day; emtricitabine 20 mg/kg/day; dolutegravir 2.5 mg/kg/day) subcutaneously (gray shaded). At time 0, (A) animal FV02 (blue diamond) received the αCEA-CAR2 control-transduced CMV-specific T lymphocytes; (B) animals FG42 (purple square) and GA75 (green triangle) received CD4-CAR2/maC46 transduced CMV-specific T lymphocytes. Plasma was collected longitudinally and assessed for viral replication by qRT-PCR.

Fig 4

Vector copy number in PBMCs and lymphoid tissues. Genomic DNA from lymphoid tissues was isolated from (A) acute PBMCs at various time points and from (B) both gut and LN at 6 weeks and PBMCs at 95 weeks after CAR2 Tc infusion in FG42, GA75 (CD4-CAR2 Tc), FV02 (CEA-CAR2), or negative control (grey) animals and then assessed for the vector-copy number by qPCR.