Broadly neutralizing antibody-derived CAR T cells reduce viral reservoir in individuals infected with HIV-1

Abstract

BACKGROUNDChimeric antigen receptor (CAR) T cells have emerged as an approach to treat malignant tumors. This strategy has also been proposed for the treatment of HIV-1 infection. We have developed a broadly neutralizing antibody-derived (bNAb-derived) CAR T cell therapy that can exert specific cytotoxic activity against HIV-1-infected cells.METHODSWe conducted an open-label trial of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of bNAb-derived CAR T cell therapy in individuals infected with HIV-1 who were undergoing analytical interruption of antiretroviral therapy (ART).RESULTSA total of 14 participants completed only a single administration of bNAb-derived CAR T cells. CAR T cell therapy administration was safe and well tolerated. Six participants discontinued ART, and viremia rebound occurred in all of them, with a 5.3-week median time. Notably, the cell-associated viral RNA and intact proviruses decreased significantly after CAR T cell treatment. Analyses of HIV-1 variants before or after CAR T cell administration suggested that CAR T cells exerted pressure on rebound viruses, resulting in a selection of viruses with less diversity and mutations against CAR T cell-mediated cytotoxicity.CONCLUSIONNo safety concerns were identified with adoptive transfer of bNAb-derived CAR T cells. They reduced viral reservoir. All the rebounds were due to preexisting or emergence of viral escape mutations.TRIAL REGISTRATIONClinicalTrials.gov (NCT03240328).FUNDINGMinistry of Science and Technology of China, National Natural Science Foundation of China, and Department of Science and Technology of Guangdong Province.

Keywords: AIDS/HIV; Clinical Trials; Immunotherapy.

Figure 1. Schematic of the clinical study.

The clinical trial was divided into 4 parts: blood drawing and CAR T cell preparation, CAR T cell infusion, ATI, and ART reinitiation after viral rebound. The safety laboratory values and HIV-1 viral load were monitored at regular intervals throughout the study.

Figure 2. Plasma viremia after discontinuation of ART in patients infected with HIV-1.

(A) Schematic representation of the lentiviral vectors carrying a gp120-specific CAR moiety containing CD28 and 4-1BB (CD137) costimulatory domains, followed by a herpes simplex virus-1 thymidine kinase (TK) and a truncated CD19 gene as the suicide genes. A combination of shRNAs, including sh-PD-1, sh-Lag-3, and sh-Tim-3, for preventing exhaustion and increasing the in vivo persistence of CAR T cells, was inserted into the vector. (B) Plasma viremia of participants in the study after the ATI of ART (n = 6). The limit of detection of HIV-1 RNA level in this assay was 20 copies/mL. (C) Kaplan-Meier curve of plasma viral suppression (< 200 copies/mL) after ATI in the trial participants.

Figure 3. Cell-associated viral RNA and in vivo CAR T cell persistence before and after adoptive transfer.

(A) Measured CAR⁺ cell concentrations (per million CD8⁺ T cells) for the 14 enrolled patients are shown in blue (log¹⁰ scale on left), and CA-RNA levels after adoptive transfer (per million CD4⁺ T cells) are shown in red (log¹⁰ scale shown on right). ATI period is shown by shades of gray. (B) HIV-1 RNA levels in plasma (copies/mL) are shown in black (log¹⁰ scale shown on right) and CA-RNA levels after ATI (per million CD4⁺ T cells) are shown in red (log¹⁰ scale shown on left). ATI periods are shown by shades of gray.

Figure 4. CAR T cell treatment decreased the CA-RNA and intact HIV-1 proviruses.

(A) Panels show the comparison of CA-RNA before and after administration of CAR T cells at indicated time points. Each point represents the mean of triplicate values. CA-RNA: 14 participants infected with HIV-1. CA-RNA (ATI): 6 ATI participants. P values were calculated using the Wilcoxon matched pairs signed-rank test. (B) Representative IPDA results from patients 002 and 015. Boxed areas are expanded to show individual positive droplets. (C) IPDA results in CD4⁺ T cells from 14 participants infected with HIV-1 (Intact proviral DNA) and 6 ATI participants (Intact proviral DNA [ATI]), before and after CAR T cell administration. P values were calculated using the Wilcoxon matched pairs signed-rank test.

Figure 5. Genetic comparison of the circulating latent reservoir and rebound viruses.

Panels show maximum likelihood phylogenetic trees of single‑genome sequencing–derived Env sequences from cell samples before CAR T cell administration and cell/plasma samples from the first and second weeks of detectable viremia. Sequences from pre-CAR T cell treatment are shown in blue, and the sequences from week 1 or week 2 of rebound viremia are shown in red. Genetic distance scale bars are shown for each tree; the bootstrap consensus trees were constructed based on HIV-1 sequences obtained from the corresponding patients. Sequences from before CAR T cell treatment were not available in patient 005.

Figure 6. Rebound virus clonality and resistance to bNAb-derived CAR T cell–mediated cytotoxicity.

(A) Clonal Env mutations on inner domain, V2 loop, loop D, CD4-binding site, and V5 loop after viral rebound in patients 002 and 006. All sequences were compared with the consensus of the rebound viruses. The residue numbers are based on HIV-1 HXB2 sequence. The top line shows amino acid differences in the pre–CAR T cell sequences from the rebound consensus. (B) PBMCs were isolated from healthy donors and divided into 2 populations. The CD8⁺ T lymphocytes were used to generate CAR T cells while the autologous CD4⁺ T cells were infected with outgrown HIV-1 from pre–CAR T cell latent reservoir (LR) or rebound reservoir (RD) (1 ng/mL p24). Six days after HIV-1 infection, antiretroviral compounds (azidothymidine and lopinavir) were added to the CD4⁺ T cell culture to inhibit virus production. Then the anti–HIV-1 drugs were withdrawn and CAR or control CD8⁺ T cells were mixed at a 1:1 ratio. Every 2 days the cultures were tested for the presence of p24 in the supernatant, using ELISA. Gray shade represents the addition of antiviral drugs. (C) HIV-1 Env derived from pre–CAR T cell latent reservoir or rebound reservoir was ectopically expressed on the HEK293T cell line. These target cell lines were compared for changes in sensitivity to CAR T cell–mediated specific cytotoxicity. Env derived from HIV-1_NL4-3 served as the positive control. Direct killing of target cell lines was tested after 24-hour coculture by detecting LDH release. A 2‑sided P value for the estimated difference in pre–CAR T cell and rebound resistance was calculated. Data represent the mean of triplicate values, and error bars represent SEM. P values were calculated using the 2-tailed unpaired Student’s t test with equal variances.