HIV-specific and resistant CAR T cells promote control of HIV replication in people with HIV

Technical Summary: HIV-specific and resistant CAR T cells promote control of HIV replication in people with HIV

Problem Statement
Contemporary antiretroviral therapy (ART) suppresses plasma viremia but fails to eradicate the latent HIV reservoir. Consequently, viral rebound is nearly universal upon treatment interruption, necessitating lifelong therapy. While immune-based strategies (e.g., broadly neutralizing antibodies, checkpoint modulation) are being explored, rapid and durable HIV-specific T-cell responses are identified as a critical determinant for post-treatment control. Previous clinical trials using HIV-specific CAR T cells (specifically CD4ζ CARs) demonstrated safety and long-term persistence but failed to consistently control viral rebound. These earlier designs had two primary limitations: the engineered cells were susceptible to HIV infection (lacking resistance), and their efficacy was not rigorously tested during structured analytical treatment interruptions (ATI). Furthermore, newer designs utilizing broadly neutralizing antibody (bNAb) domains showed reservoir reduction but did not achieve durable functional cure or consistent viremia control after ART cessation.

Methodology
This first-in-human pilot study (NCT03617198) evaluated autologous, engineered T cells designed to be both HIV-specific and HIV-resistant.

• Cell Engineering: T cells were harvested via leukapheresis from ten men with well-controlled HIV. The cells were modified using a multi-step process:
  1. CCR5 Editing: T cells were electroporated with CCR5-specific zinc-finger nucleases (ZFNs) to disrupt the CCR5 co-receptor, rendering them resistant to R5-tropic HIV infection.
  2. CAR Transduction: Cells were transduced with a third-generation lentiviral vector encoding a chimeric antigen receptor (CAR). This CAR utilized the extracellular domain of CD4 to bind the HIV envelope (Env), coupled with 4-1BB and CD3ζ signaling domains to enhance persistence and function.
  3. Manufacturing: The final product (CCR5-edited CD4BBζ CARTs) was cultured for at least 9 days.
• Clinical Protocol: Participants received a single infusion of up to 10 billion engineered T cells. The study employed a two-cohort design to assess the impact of post-infusion maturation:
  • Cohort 1: ATI initiated 1 day post-infusion.
  • Cohort 2: ATI initiated at least 8 weeks post-infusion.
  • The ATI lasted 16 weeks, with safety triggers for ART re-initiation. Participants with viral loads ≤1,000 copies/mL could extend the ATI.
• Assessments: The study monitored safety, CART persistence, viral rebound kinetics, reservoir size (via Intact Proviral DNA Assay [IPDA] and WPRE quantification), proviral composition (FLIP-seq), integration sites (MIP-seq), and endogenous HIV-specific CD8 T-cell responses (ELISpot).

Key Results

• Safety: The infusion was well-tolerated with no serious adverse events, cytokine release syndrome (CRS), or neurotoxicity.
• Viral Rebound and Control:
  • Viral rebound occurred in 9 of 10 participants within 1–6 weeks. One participant (Participant 05) showed no detectable rebound during the 16-week ATI.
  • Post-Rebound Control: Six participants (01, 02, 06, 08, 10, 12) achieved viral loads below their pre-ART set points after rebound.
  • Durable Control: Participant 02 maintained plasma HIV RNA below 1,000 copies/mL for 90 weeks without ART. Participant 06 exhibited a rapid decline in viral load (from ~325,000 to 105 copies/mL) during the ATI.
  • Expansion Dynamics: Unlike CD19-CARs, HIV-specific CARTs did not show massive peripheral expansion in most participants; levels generally declined over time. However, Participant 05 showed a remarkable expansion of CARTs 43 days after ATI initiation despite no detectable viremia, suggesting recognition of low-level viral recrudescence.
• Reservoir Analysis:
  • IPDA results initially appeared to show an increase in intact proviral DNA. However, after correcting for the lentiviral vector sequences (WPRE) present in the CARTs, no statistically significant reduction in the intact reservoir was observed.
  • FLIP-seq and MIP-seq revealed no consistent changes in reservoir size, clonal composition, or integration site distribution. However, participants with control (02, 05, 08) showed enrichment of intact proviruses in heterochromatin regions.
• Immune Reinvigoration: CART infusion was associated with a broad reinvigoration of endogenous HIV-specific CD8 T-cell responses. Participant 02, who maintained the longest control, showed the largest increase in Gag-specific CD8 T-cell reactivity.
• Immune Escape: In Participant 08, sequencing revealed a mutation in the Gag protein (Glycine to Glutamine at position 359) that emerged during the ATI. Functional assays confirmed this mutation abolished T-cell recognition, providing direct evidence that the enhanced immune response exerted selective pressure on the virus.

Significance and Claims
The authors claim that this study provides the first clinical evidence that HIV-specific, HIV-resistant CAR T cells can alter viral dynamics following ART withdrawal. The findings suggest that these engineered cells can:

1. Mediate Virologic Control: In a subset of individuals, CARTs can prevent viral rebound or suppress replication to levels below the historical set point after rebound.
2. Enhance Endogenous Immunity: The therapy acts not only through direct killing of infected cells but also by reinvigorating natural HIV-specific CD8 T-cell responses, which can drive immune escape mutations.
3. Safety and Persistence: The approach is safe and the engineered cells can persist long-term without clonal expansion or transformation.

The paper concludes that while the study did not demonstrate a reduction in the latent reservoir size, the observed post-rebound control supports a model where HIV-specific CARTs contribute to a "functional cure" by containing viral replication. The authors posit that the timing of ATI relative to infusion (allowing for engraftment/maturation) and the degree of HIV resistance (e.g., CCR5 editing efficiency) are critical factors for efficacy. They suggest that HIV-specific CARTs may serve as an integral component of future HIV cure strategies, potentially in combination with other interventions.