Research ArticleHIV

Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model

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Science Translational Medicine  07 Aug 2019:
Vol. 11, Issue 504, eaav5685
DOI: 10.1126/scitranslmed.aav5685
  • Fig. 1 Illustration of the multispecific anti-HIV CAR architecture.

    (A) Gene structure of multispecific anti-HIV CARs encoded by LVs. The term monospecific, bispecific, or trispecific refers to the total number of binding specificities contained in the CAR molecule(s) within a single construct. For simplicity, each anti-HIV binder is assigned a number. For instance, mD1.22 = 1, m36.4 = 3, and C46 peptide = 4. The term monoCAR (M; one-molecule CAR architecture) or duoCAR (D; two-molecule CAR architecture) refers to the number of CAR molecules encoded by an LV and expressed in a single cell. For instance, a monoCAR containing the mD1.22 domain is referred to as M1, or a duoCAR containing the mD1.22 and m36.4 domains is called D13. An illustration of the Env gp120/gp41 trimer and putative target sites for each anti-HIV binder is shown on the right. (B) Cartoon illustration of each anti-HIV CAR used in the study. Monospecific CARs contain either mD1.22 (1) or m36.4 (3) fused to a CD8 ectodomain (EC), CD8 transmembrane domain (TM), 4-1BB costimulatory domain, and CD3ζ T cell signaling chain to form M1 or M3, respectively. The conventional bispecific CAR contains mD1.22 fused to m36.4 via a 3xG4S motif to form a one-molecule CAR construct (M13). The bispecific duoCAR contains the mD1.22-CAR and m36.4-CAR coexpressed in T cells using a two-molecule architecture (D13). In another iteration, the D134Δ construct contains the bispecific CAR (M13) in addition to the C46 peptide that is anchored to the T cell membrane but does not contain a CD3ζ T cell signaling chain (4Δ). Last, the trispecific duoCARs contain three specificities across two CAR molecules within a single cell. The first CAR contains the C46 peptide (4) fused to the N terminus of the mD1.22 (1) domain via a 3xG4S or 5xG4S linker (designated “S” for short or “L” for long) and a second CAR containing the m36.4 domain (3) to form D413S or D413L, respectively.

  • Fig. 2 LV-encoded multispecific anti-HIV CARs expressed on the surface of CD4+ and CD8+ T cells capture HIV Env.

    (A) Representative detection of anti-HIV CARs via the mD1.22 domain on the surface of activated primary T cells using anti-CD4 flow cytometry after transduction and expansion (n = 7 donors). VioBlue-conjugated CD4 antibody recognizes the D1 domain of the native CD4 receptor expressed by CD4+ T cells and the mD1.22-CAR, which contains a modified D1 domain. UTD T cells are used as a biological control to set gates. The x axis shows CD4+ T cells (Q4) and mD1.22 expression on CD4+ T cells in the quadrant labeled CAR+ CD4+. The y axis shows CD8+ T cells (Q1). mD1.22 expression on CD8+ T cells is shown in the quadrant labeled CAR+ CD8+. FITC, fluorescein isothiocyanate. (B) Representative detection of trispecific CARs via the C46 peptide using 2F5 Mab directed against the gp41 MPER region (n = 5 donors). Graphical representation of % CAR-modified T cells from multiple donors using either (C) anti-CD4 (n = 7 donors) or (D) anti-C46 (2F5 Mab) flow cytometry (n = 5 donors). The error bars represent mean ± SD of independent donors. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Bonferroni posttest analysis (***P < 0.0001 and ****P < 0.00001). IgG, immunoglobulin G; PE, phycoerythrin; FACS, fluorescence-activated cell sorting. (E) Measurement of soluble HIV envelope (Env) capture by all T cells. Representative histogram is shown (n = 2 donors). Specific binding of anti-HIV CARs to soluble, His-tagged gp140 (clade B) relative to the endogenous CD4 receptor expressed on CD4+ T cells in the total T cell population. UTD T cell control and CD19-CAR-T cells serve as a control for specificity. (F) Measurement of soluble HIV envelope capture by CD8+ T cells. Representative histogram is shown (n = 2 donors).

  • Fig. 3 Cytotoxicity of anti-HIV CAR-T cells against HIV-1 Env-expressing target cells.

    (A) BnAb detection of the HIV-1 Env glycoprotein on the surface of cell lines used for cytotoxicity assays. (B) Cytotoxicity of conventional CD4-CAR versus mD1.22-CAR-T cells against Env+ target cells (representative figure, n = 2 donors) and (C) Env target cells (293T-Luc). The mCherry reporter–modified donor T cells serve as a negative control. (D) Cytotoxicity of monospecific and bispecific anti-HIV CAR-T cells against Env+ and Env target cells, (E) 293T-Luc, and (F) Raji-Luc. (G) IFN-γ secretion from monospecific and bispecific anti-HIV CAR-T cells upon challenge with Env+ target cells. (H) Cytotoxicity of multispecific anti-HIV CAR-T cells against Env+ and Env target cells, (I) 293T-Luc, and (J) Raji-Luc. (K) IFN-γ secretion from multispecific anti-HIV CAR-T cells in the presence of Env+ target cells. n = 3 donors for monospecific versus bispecific CARs; n = 2 donors for bispecific versus trispecific CARs. The error bars represent ±SEM. Statistical analyses were performed by two-way ANOVA followed by Bonferroni posttest analysis performed for all comparisons (***P < 0.0001, **P < 0.001, and *P < 0.01; n.s., not significant).

  • Fig. 4 Anti-HIV duoCAR strongly induces T cell activation without impairing functionality.

    (A) Expression profile of the anti-HIV CARs used to determine CD107a and cytokine production upon challenge with HIV-infected donor-matched PBMCs. (B) CAR-T cell activation profile 6 hours after challenge with HIV-infected PBMCs. The graph shows the percentage of UTD T cell control or CAR-T cells producing the indicated T cell activation marker (CD107a) or intracellular cytokine (IL-2, IFN-γ, or double-positive for IL-2 and IFN-γ) in the presence of donor-matched PBMCs infected with Du422.1-IMC-LucR virus. The graph shows pooled data from combined triplicate wells of a single donor. (C) Exhaustion marker profile of anti-HIV CAR-T cells in the absence of antigen stimulation. The coexpression of Lag-3, PD-1, and Tim-3 on the surface of anti-HIV CAR-T cells in the absence of antigen stimulation was quantified by flow cytometry and analyzed in SPICE 6 software. The pie slices show the proportion of cells expressing 0, 1, 2, or 3 inhibitory receptors. The arc above the pie slice indicates the inhibitory receptor(s) expressed by effector T cells. Representative data are shown (n = 2 donors).

  • Fig. 5 Broad and potent in vitro killing of HIV-infected PBMCs by multispecific duoCAR-T cells.

    (A) Schematic of the in vitro HIV-LucR PBMC killing assay. IMCs of HIV-1 encoding a heterologous HIV-1 Env glycoprotein and a Renilla luciferase cassette (generically referred to as Env-IMC-LucR or HIV-LucR) were used to infect donor-matched PBMCs followed by coculture with UTD T cell control or CAR-T cells for 7 days. After 7 days, the cocultures were lysed, and luciferase activity was assessed to quantify HIV-1 infection. (B) In vitro HIV challenge of anti-HIV CAR-T cells with PBMCs infected with Du422.1-IMC-LucR virus (VRC01/3BNC117-resistant Env, clade C). T, targets (Du422.1-IMC-LucR–infected PBMCs). Data shown are ±SD of triplicate wells for four different donors. Statistical analysis was performed using a pairwise Student’s t test for each donor. Significance is considered P < 0.05. (C) In vitro HIV challenge of anti-HIV CAR-T cells with PBMCs infected with Env-IMC-LucR viruses expressing Env found worldwide. The figure shows averaged log HIV-1 inhibition relative to UTD T cells (n = at least 3 donors tested in triplicate; n = 2 donors tested in triplicate for AE.CNE55). Log inhibition of HIV-1 infection was calculated by the following formula using background-corrected relative light units (RLUs) obtained for each sample: log inhibition = log10(RLUCAR/RLUUTD) and then averaged across donors. Statistical analysis was performed by pairwise comparison of CAR constructs across multiple donors via a fixed-effects ANOVA model of background-corrected log inhibition of HIV-1 infection adjusting for donor and treatment. To account for multiple comparisons, we chose a more conservative P value of ≤0.01 for statistical significance.

  • Fig. 6 Multispecific anti-HIV CAR-T cells are protected from HIV-1 infection.

    CD4-enriched anti-HIV CAR-T cells were directly challenged with Env-IMC-LucR viruses encoding env from strains (A) BaL (clade B, R5-tropic), (B) NL4-3 (clade B, X4-tropic), (C) SF162 (clade B, R5-tropic), (D) CAP45 (clade C, partially resistant to VRC01), (E) C.Du172.17 (clade C, resistant to VRC01), or (F) C.Du422.1 (clade C, resistant to VRC01/3BNC117). Anti-HIV CARs were also challenged with Env-IMC-LucR viruses expressing Env from representative HIV-1 clade AC (G) AC.246-F3, clade AE (H) AE.CNE8, a second clade AE (I) AE.CNE55, clade BC (J) BC.CH119.10 (partially resistant to VRC01), and clade G (K) GX1632_S2_B10. Donor-matched HIV-negative PBMCs (HIV PBMC) and HIV-infected PBMCs (HIV+ PBMC) serve as negative and positive controls for the assay, respectively. The error bars shown are ±SD of three independent donors tested in triplicate. Statistical analysis was performed by pairwise comparison of CAR constructs across multiple donors via a fixed-effects ANOVA model of log-transformed RLU values adjusting for donor and treatment. Significance is considered P ≤ 0.01.

  • Fig. 7 Anti-HIV duoCAR-T cells are more potent than monoCAR-T cells in suppressing bNAb-resistant HIV infection in vivo.

    (A) Illustration of the hu-spl-PBMC-NSG mouse model of intrasplenic HIV infection and experimental design for acute HIV infection. (B) In vivo efficacy of monoCAR-T cells versus duoCAR-T cells against donor-matched PBMCs infected with C.Du422.1-IMC-LucR virus. The relative % HIV inhibition was calculated using the Renilla luciferase activity detected in 1 × 106 splenocytes by the formula: [1 − (RLUCAR/RLUUTD)] × 100. The relative % HIV-1 inhibition is indicated on the bar graph above the corresponding CAR-T group. The overall and pairwise group differences in log-transformed values were evaluated via ANOVA and Dunnett’s test for statistical analysis. (C) Percentage of human CD4+ T cells detected in the spleens of HIV-infected mice by flow cytometry during acute HIV infection. (D) Percentage of human CD8+ T cells detected in the spleens of HIV-infected mice by flow cytometry during acute HIV infection. (E) Quantitative polymerase chain reaction (qPCR) detection of CAR-T cells in the spleens of HIV-infected mice during acute infection. The plots show n = 6 mice per group except for the HIV-negative PBMC control group (HIV-neg PBMCs), which is n = 4 mice. One of the mice in the M1 CAR–treated group did not have enough cells for the analyses in Fig. 7, C to E. The experiment was performed once with a single donor. The error bars represent ±SD.

  • Fig. 8 Multispecific anti-HIV duoCAR-T cells display potent in vivo HIV-1 suppression and mitigate CD4+ T cell loss during persistent infection.

    In vivo efficacy of bispecific and trispecific anti-HIV duoCAR-T cells against PBMCs infected with C.Du422.1-IMC virus after (A) 7 days and (B) 30 days of HIV-1 infection. Mice were intrasplenically injected with 5 × 106 total CAR-T cells and 1 × 107 HIV-infected PBMCs on day 0. (C and D) Percentage of human CD4+ T cells detected by flow cytometry in the spleens of HIV-infected mice treated with UTD T cell control or duoCAR-T cells after (C) 7 days and (D) 30 days of HIV-1 infection. (E and F) Percentage of human CD8+ T cells detected by flow cytometry in the spleens of HIV-1 infected mice treated with UTD T cell control or duoCAR-T cells after (E) 7 days and (F) 30 days of HIV-1 infection. (G and H) Detection of duoCAR-T cells in HIV-infected spleens after (G) 7 days and (H) 30 days of HIV-1 infection. DuoCAR-T cells were detected in the spleens of HIV-infected cohorts by cFrag qPCR. For the 7-day HIV study, two different donors were used to generate CAR-T cells. The error bars represent ±SD (n = 9 mice for HIV-infected groups treated with UTD T cell control or CAR-T cells; n = 4 mice for the HIV-neg PBMC group). For the 30-day HIV infection study, the experiment was performed using one donor to generate CAR-T cells. The error bars represent ±SD (n = 5 mice for HIV-infected groups treated with UTD T cell control or CAR-T cells; n = 2 mice for the HIV-negative PBMC group). Statistical analysis was performed by one-way ANOVA followed by Tukey’s posttest analysis. Significance is considered P < 0.05, and only significant P values are plotted.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/504/eaav5685/DC1

    Materials and Methods

    Fig. S1. Flow cytometry gating strategy for detection of anti-HIV CARs on the surface of CD4+ and CD8+ T cells.

    Fig. S2. Ratio of CD4+ and CD8+ T cells for different donors used in the study.

    Fig. S3. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from HIV-1 clade B viruses.

    Fig. S4. Multispecific anti-HIV CAR-T cells exhibit superior in vitro killing efficacy at low E:T ratios.

    Fig. S5. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from two HIV-1 clade C viruses.

    Fig. S6. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from HIV-1 clades AC, BC, and G viruses.

    Fig. S7. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from HIV-1 clade AE virus.

    Fig. S8. Percentage of HIV-1 inhibition for conventional and multispecific anti-HIV CAR-T cells tested against PBMCs infected with 11 different Env-IMC-LucR viruses encoding genetically diverse env genes.

    Fig. S9. In vitro elimination by anti-HIV CAR-T cells of PBMCs infected with an IMC expressing Env from HIV-1 clade C virus.

    Fig. S10. Simultaneous expression of the mD1.22 and m36.4 domains on the surface of mono- and duoCAR-T cells.

    Fig. S11. Detection of total cell-associated HIV DNA in the spleens of HIV-infected NSG mice treated with mono- and duoCAR-T cells.

    Data file S1. Primary data.

    References (55, 56)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Flow cytometry gating strategy for detection of anti-HIV CARs on the surface of CD4+ and CD8+ T cells.
    • Fig. S2. Ratio of CD4+ and CD8+ T cells for different donors used in the study.
    • Fig. S3. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from HIV-1 clade B viruses.
    • Fig. S4. Multispecific anti-HIV CAR-T cells exhibit superior in vitro killing efficacy at low E:T ratios.
    • Fig. S5. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from two HIV-1 clade C viruses.
    • Fig. S6. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from HIV-1 clades AC, BC, and G viruses.
    • Fig. S7. In vitro killing efficacy of multispecific anti-HIV CAR-T cells against PBMCs infected with an IMC expressing Env from HIV-1 clade AE virus.
    • Fig. S8. Percentage of HIV-1 inhibition for conventional and multispecific anti-HIV CAR-T cells tested against PBMCs infected with 11 different Env-IMC-LucR viruses encoding genetically diverse env genes.
    • Fig. S9. In vitro elimination by anti-HIV CAR-T cells of PBMCs infected with an IMC expressing Env from HIV-1 clade C virus.
    • Fig. S10. Simultaneous expression of the mD1.22 and m36.4 domains on the surface of mono- and duoCAR-T cells.
    • Fig. S11. Detection of total cell-associated HIV DNA in the spleens of HIV-infected NSG mice treated with mono- and duoCAR-T cells.
    • References (55, 56)

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    Other Supplementary Material for this manuscript includes the following:

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