Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity.

Gammaretrovirus vector production and transduction of T cells.

The gammaretroviruses carrying the CAR transgenes were made by transient transfection as described previously (56). Briefly, 293GP cells (BD Biosciences) were cotransfected with retroviral vector plasmid and the plasmid encoding RD114 envelope protein using Lipofectamine 2000 reagent (Life Technologies). Supernatants containing retrovirus were collected at 48 h posttransfection and stored at −80°C.

For transduction, non-tissue culture-treated 6-well plates were coated with 1.5 ml/well RetroNectin (TaKaRa) (10 μg/ml, diluted 1:100 in phosphate-buffered saline [PBS]) at 4°C overnight. The following day, retroviral supernatant was rapidly thawed and diluted 1:1 in serum-free RPMI 1640 medium. A 4-ml volume of retroviral supernatant (diluted 1:1) was added per well to RetroNectin-coated plates, which were then centrifuged at 2,000 × g for 2 h at 32°C. The supernatant was then aspirated from the wells, and 1.5 ml of activated PBMCs was added per well at a density of 0.5 × 10⁶ cells/ml in T cell medium containing IL-2. The plates were then centrifuged for 10 min at 1,000 × g and incubated at 37°C overnight. The next day, the transduced PBMCs were given a second round of transduction following the same procedure. The cells were then cultured at 37°C in 5% CO₂ until analysis for CAR expression was performed.

Detection of CARs on transduced T cells.

As recommended by BD Biosciences, approximately 1 × 10⁶ cells were washed, suspended in fluorescence-activated cell sorter (FACS) buffer (PBS [pH 7.4] plus 0.4% bovine serum albumin [BSA] and 0.1% sodium azide), and incubated for 30 min at 4°C with the following reagents: for detecting CD4-based CARs, fluorescein isothiocyanate (FITC)-labeled anti-CD3 antibody (clone HIT3a), allophycocyanin (APC)-labeled anti-CD8 antibody (clone SK1), and phycoerythrin (PE)-labeled anti-CD4 antibody (clone RPA-T4); for detecting the 139 CAR, protein l-biotin followed by PE-streptavidin. After washing in FACS buffer was performed, flow cytometry acquisition was performed with a BD FACSCalibur or FACSCanto II system (BD Biosciences), and data analysis was performed with FlowJo (Treestar)

(Fig. 2).

FIG 2

Detection of CARs on transduced T cells. CD3⁺ cells (FITC-labeled anti-CD3 antibody, clone HIT3a) were gated on CD8⁺ APC-labeled anti-CD8 antibody (clone SK1). The CD4 CAR (top four panels) was detected with PE-labeled anti-CD4 antibody (clone RPA-T4); the 139 CAR was detected with protein l-biotin followed by PE-streptavidin. Data on the x and y axes are based on relative fluorescence intensities (arbitrary units).

Regulated expression of HIV Env in HeLa Tet-Off cells.

We employed the Tet-Off system to regulate Env expression in target cells (53).. For Western blot analysis of Env expression Fig. 3A, HeLa Tet-Off cells (ClonTech) were transiently transfected with plasmid pGL4.22-JRFL and incubated for 2 days in the presence of the indicated concentrations of doxycycline. Lysate samples (from 3 × 10⁵ cells) were resolved under reducing conditions on a 4% to 20% gel using SDS-MOPS (SDS-morpholinepropanesulfonic acid) as the running buffer; a lysate from the same number of CHO-env cells served as a positive control. After transfer, the Western blot membrane was first subjected to a reaction with a 1:800 dilution of a polyclonal sheep anti-gp120 antibody (D7324; Aalto Bio Reagents) followed by incubation with rabbit anti-sheep IgG conjugated to horseradish peroxidase (HRP; Sigma). SuperSignal West Dura extended-duration substrate (Thermo Scientific) was used for final detection.

FIG 3

Regulated expression of JR-FL Env and its effect on activation of CAR-transduced PBMCs. (A) Western blot analysis of whole-cell lysates of HeLa Tet-Off cells showing the level of JR-FL Env expression in the presence of various concentrations of doxycycline. A lysate from untransfected cells (UT) served as a negative control; CHO-env cells, stably expressing HIV-1 III_B Env, served as a positive control. Only the relevant region of the blot is shown. (B) IFN-γ production in cocultures of Env-expressing HeLa target cells and CAR-transduced effector PBMCs. Three populations of target cells were tested that had been prepared by growing HeLa Tet-Off cells in the presence of 0, 2, or 20 ng/ml of doxycycline. The effector cells expressing the 139 CAR and untransfected (UT; Env-negative) target cells served as negative controls. The error bars represent standard deviations of the results derived from duplicate samples.

For analysis of Env activation of CAR-transduced T cells, HeLa Tet-Off cells were seeded at 1 × 10⁴ per well in a 96-well plate overnight and then transfected with plasmid pGL4.22-JRFL using FugeneHD (Promega) according to the manufacturer's protocol and incubated in the absence or presence of the indicated concentrations of doxycycline. After 2 days, medium was aspirated from each well and replaced with 100 μl fresh medium containing 45 × 10³ effector cells. Negative controls were prepared by using untransduced effector cells or effector cells expressing the 139 CAR. The plate was incubated overnight, and the media were diluted 1:5 the following day for a gamma interferon (IFN-γ) enzyme-linked immunosorbent assay (ELISA) (Thermo kit; catalog no. EHIFNG) (Fig. 3B).

Cytotoxicity assay.

Direct cytotoxicity assays ((Fig. 4A) were performed using a radioisotope-free luminescence-based CytoTox-Glo kit (Promega) following the manufacturer's protocol to measure protease activity released from dead cells. Briefly, triplicate experiments using serial 2-fold dilutions of effector CAR-transduced T cells and a 96-well plate were performed, with the highest cell number being 1.87 × 10⁵ per well in 50 μl. Equal volumes of target cell suspension, containing 1.87 × 10⁴ cells, were added to all wells, and the cells were cocultured for 4 h at 37°C in 5% CO₂. CytoTox-Glo reagent was then added to each well followed by brief mixing on an orbital shaker and then incubation at room temperature for 15 min. Luminescence was measured using a Bio-Tek luminometer. Luminescence values determined from wells containing effector cells alone and target cells alone were combined and subtracted as the background from the values determined from the cocultures. The contents of wells containing target cells alone were mixed with a lysis reagent for 15 min at room temperature, and the resulting luminescence was considered to represent 100% lysis.

FIG 4

Targeted killing of Env-expressing cells by CD4-based CARs. (A) Direct killing of target cell lines. Direct cytotoxicity assays were performed using a CytoTox-Glo kit and, as targets (T), CHO, the CHO-env stable transfectant, and Raji cells. Cocultures were performed for 4 h with T cells expressing the indicated CARs (effector cells [E]); the 139 CAR served as a negative control. Error bars (representing negligible values) indicate standard deviations of the results from triplicate samples. (B) Suppression of HIV-1 pseudovirus production from producer target cells. HEK293T target cells (T) transfected with plasmids directing production of HIV-1 infectious pseudovirus particles (Env from the QH0692 primary isolate) were cocultured with CAR-transduced PBMCs (effector cells [E]) at the indicated effector/target (E:T) ratios. The amounts of infectious pseudovirus released into the supernatants were quantified by incubation with SupT1-DCSIGNR cells. Pseudovirus production in the absence of effector cells was defined as 100%. Untransduced effector cells served as a negative control. Error bars indicate standard deviations of the results from quadruplicate samples. For each panel, the number of target cells is defined as 1.

CAR-mediated inhibition of HIV-1 pseudovirus production.

HEK293T cells were transfected with plasmids to generate the luciferase gene-carrying pseudovirus (Env from QH0692 primary isolate) as described previously (57). At 6 h posttransfection, 1 × 10⁵ transfected cells (target cells) were mixed with CAR-transduced PBMCs (effector cells) at the indicated effector/target ratios. Cells were cocultured at 37°C for 2 days to allow pseudovirus release from target cells into the culture supernatant. Coculture supernatants were then collected, cleared by centrifugation at 2,000 rpm for 5 min, and assayed for infectious pseudovirus content. Pseudovirus infections of SupT1-DCSIGNR cells were performed in 96-well round-bottom plates in which 25 μl of pseudovirus-containing cleared culture supernatant was inoculated onto 2 × 10⁵ cells per well in quadruplicate in the presence of 20 μg/ml of DEAE-dextran. At 48 h postinfection, infected cells were analyzed for luciferase content as previously described (57).

Inhibition of primary spreading HIV-1 infection by CAR-T cells.

Frozen autologous PBMCs were rapidly thawed and suspended at a density of 2 × 10⁶/ml in RPMI 1640 medium containing 20% FBS, IL-2, and 50 μg/ml phytohemagglutinin (PHA). A 2-ml volume of cell suspension was added to each well of a 24-well plate and incubated in 5% CO₂ at 37°C overnight. The next day, cells were collected and resuspended with fresh medium without PHA. After 2 to 3 days of culture, cells were resuspended at 5 × 10⁶/ml in RPMI 1640 medium (containing 20% FBS and IL-2) and transferred to a T25 flask. A 1-ml volume of primary HIV-1 isolate stock (p24 titer of 50 to 150 ng/ml) was then added to each flask, and the cells were incubated at 37°C in 5% CO₂ overnight. Infected cells were spun down at 300 × g for 10 min. After removal of the supernatant, cells were washed 3 times using 20 ml of medium per wash and then resuspended in complete medium (RPMI 1640, 20% FBS, and IL-2) at a density of 1.5 × 10⁶ cells per ml in 96-well round bottom plates. Subsequently, 100 μl of infected PBMCs (targets [T]) was mixed in duplicate with 100 μl of serially diluted CAR-transduced T cells (effectors [generated from the same donor]) to obtain the various effector/target ratios indicated in the figures. Cocultures were performed at 37°C in 5% CO₂ for 8 days, at which time supernatants were collected and assayed for p24 content by ELISA (PerkinElmer). For comparisons of the relative CAR activities using cells from different donors (Fig. 5A), autologous PBMCs from 3 different healthy donors were used to generate HIV-1 (BX08 isolate)-infected target and CAR-transduced effector cells. For analysis of relative CAR activities using different HIV-1 isolates (Fig. 5B), single-donor autologous PBMCs were used to generate target and effector cells for coculture.

FIG 5

Suppression of spreading HIV-1 infection in PBMCs by T cells expressing various CD4-based CARs. (A) BX08 (clade B) data show the average level of infection of PBMCs from multiple donors, normalized to an infection obtained with effectors expressing the 139 CAR at a 0.04:1 E:T cell ratio (defined as 100). (B) CD4-based CARs were tested against the indicated HIV-1 isolates: CAM0015BBY, clade CRF02_AG; JR-FL and AD8, clade B; and RW020 and KNH1135, clade A. The 139 CAR was used as a negative control. In cases where points fell below detectable levels, horizontal dotted lines are shown to indicate the limits of detection.

Epitope exposure on pseudovirus captured by HOS cells expressing CD4-based CARs.

Human osteosarcoma (HOS) cells were transduced to express CD4-based or control CARs using the method described for human PBMC transduction; untransduced cells provided a negative control. CD4 CAR expression on HOS cells was measured with PE-labeled anti-CD4 (clone RPA-T4) following the method described for CAR detection on transduced T cells; equivalent expression levels were observed for all the CD4-based CARs (not shown). A total of 1 × 10⁶ CD4-based CAR-expressing HOS cells were washed and suspended in 100 μl containing HIV-1 pseudovirus (Ba-L) for 30 min at 37°C; an equivalent incubation in the absence of pseudovirus served as a negative control. Cells were then washed three times with FACS buffer and incubated with either 2G12 or 17b human MAb for 30 min at 4°C. Cells were washed again three times and incubated with a goat anti-human (Hu)-IgG Alexa Fluor 488 conjugate. Flow cytometry acquisition was performed with a BD FACSCalibur system (BD Biosciences), and analysis was performed with FlowJo (Treestar) (Fig. 6).

FIG 6

Accessibility of the 17b moiety on HIV-1 pseudovirions captured by HOS (CD4-negative) cells expressing the indicated CARs. Flow cytometry was performed to detect binding of anti-gp120 MAbs on captured virions; MAb 2G12 binding served as a positive control for virion capture; MAb 17b revealed accessibility to the CD4-induced 17b epitope. HOS cells without pseudovirions served as a control for staining specificity. Untransfected cells (UT) served as an additional negative control. Data on the x axes are based on relative fluorescence intensities (arbitrary units).

Susceptibility of HOS-CCR5 cells expressing CD4-based CARs to pseudovirus infection.

HOS-CCR5 cells were transduced with retroviruses encoding the various CD4-based CARs; their surface expression data were confirmed to be comparable by flow cytometry (>70% CAR positive). Equal numbers (10⁴) of each transduced cell population were seeded overnight in quadruplicate in 96-well plates followed by incubation with 4-fold serial dilutions of Ba-L pseudovirus in the presence of 20 μg/ml DEAE-dextran. After 48 h, the media were aspirated and the cell lysates were assayed for luciferase activity as described previously (57) (Fig..7A).

FIG 7

HIV-1 infection susceptibility of cells expressing CD4-based CAR variants. (A) Pseudovirus infectivity (Env from the Ba-L isolate) of HOS-CCR5 cells expressing CD4-based CARs. The error bars represent standard deviations of the results from quadruplicate samples. The extremely low relative light unit (RLU) values for both CD4-17b CARs and the CD4-35-DDY3 CAR were indistinguishable from background values as defined by value determined from the HOS-CCR5 negative-control cells (approximately 15 RLU). (B) Susceptibility of CD8⁺ T cells expressing the different CD4-based CARs to infection by cell-free HIV-1. CD8⁺ T cells obtained by negative selection and expressing the indicated CARs were challenged with cell-free HIV-1. Infection was analyzed by flow cytometry and staining for intracellular p24. (Upper panel) The CD8⁺ T cells were challenged with the Ba-L isolate and analyzed 3 days later. Untransduced (CAR-negative) cells served as a negative control. The percent value in each dot blot indicates the p24-positive population. Data on the x axes are based on relative fluorescence intensities (arbitrary units). (Lower panel) CAR-expressing CD8⁺ T cells were challenged with the AD8 isolate. Infection was assessed by the increase in intracellular p24 staining at h 0 (background) compared to h 72.

CAR expression on transduced PBMC-derived T cells.

PBMCs from a healthy donor were stimulated for 2 days with IL-2 and the anti-CD3 MAb OKT3; the resulting T cell-enriched population was then transduced (for 2 cycles) with pMSGV-1 gammaretroviral vectors encoding the designated CARs. Samples were analyzed by flow cytometry at 5 days after the second transduction. As shown in Fig. 2, transduction efficiencies were high for all CD4-based CARs as well as for the control 139 CAR (55). In multiple experiments with PBMCs from different donors, the transduction efficiencies typically were >70% and were similar for all CARs used in a given experiment.

HIV-1 Env stimulation of IFN-γ release from CAR-transduced T cells.

Target cells transiently expressing various levels of HIV-1 Env (JR-FL isolate) were generated by transfecting HeLa Tet-Off cells with plasmid pGL4.22-JRFL encoding Env (53) and by culturing for 48 h in the presence of the indicated levels of doxycycline (a tetracycline analog that represses Env transcription by binding to the tetracycline transactivator and preventing its interaction with the upstream tetracycline response elements); untransfected cells served as negative controls. Western blot analyses (Fig. 3A) indicated high-level Env expression in the absence of doxycycline and marked suppression in response to increasing doxycycline concentrations (0.2 to 20 ng/ml); at much longer (overnight) exposure, only very faint gp120 bands were observed, with equivalent intensities for 20 and 2,000 ng/ml doxycycline (not shown), presumably reflecting “leakiness” of the Tet-Off system. Figure 3B shows that transduced effector T cells expressing all three CD4-based CARs were stimulated to secrete IFN-γ in specific response to coculture with Env-expressing target cells but not with control untransfected target cells lacking Env; further specificity was demonstrated by the absence of IFN-γ secretion with control effector cells expressing the 139 CAR. The potent sensitivity of the specific effector cell response is illustrated by the relatively high level of IFN-γ secreted (Fig. 3B; ∼40% to ∼60% of maximum), with target cells expressing extremely barely detectable Env levels (Fig. 3A) under conditions of suppression by high-dose (20 ng/ml) doxycycline.

Specific killing of Env-expressing cells by CD4-based CARs.

We tested the CAR-transduced T cells for specific direct cytotoxic activity against Env-expressing target cells using a CytoTox-Glo kit (Fig. 4A). All three CD4-based CARs displayed robust cytotoxicity against the CHO-env cell line constitutively expressing Env from the T cell line-adapted IIIB isolate, at comparable efficacies over the indicated range of effector/target ratios. Specificity was confirmed by the absence of killing of the Env-negative parental CHO cells and by the lack of activity of effector cells transduced with the control 139 CAR. Importantly, the CD4-based CARs displayed no cytotoxicity against Raji cells, which express very high levels of major histocompatibility complex (MHC) class II (MHC-II) molecules (62), the natural binding partner for CD4; this finding parallels previous demonstrations of the absence of activity of CD4 CARs against MHC-II (35, 39) and presumably reflects the weak affinity of CD4 for MHC class II (63).

As an additional test of specific CAR-mediated cytotoxic activity, we analyzed suppression of release of infectious pseudovirus particles from 293T producer cells. This system enabled analysis of activity against the recombinant Env of choice. As shown in Fig. 4B with Env from the QH0692.42 primary isolate, all three CD4-based CARs strongly suppressed pseudovirus production over the indicated effector/target range, whereas the 139 control CAR had no effect.

Suppression of spreading HIV-1 infection in PBMCs.

The CARs were tested against spreading HIV-1 infection in PBMCs, initiated by the input of cell-free HIV-1 and assay of supernatant p24 at day 8 postinfection (Fig. 5). In each assay, PBMCs from the same healthy donor were used as target cells for infection and to generate CAR-transduced T cells. Figure 5A summarizes data from experiments in which PBMCs from three different healthy donors were infected with the BX08 primary isolate (R5) and cocultured with T cells expressing the indicated CARs at various E:T ratios; p24 levels are expressed relative to cocultures with the control 139 CAR at the lowest E:T ratio (defined as 100). The small error bars indicate that comparable patterns were observed with PBMCs from all three donors. The results demonstrate various levels of suppressive activities by the three CD4-based CARs (CD4-10-17b > CD4 > CD4-35-17b); the control 139 CAR was ineffective. These analyses were extended to multiple HIV-1 primary isolates (all R5) representing different genetic subtypes, each tested with PBMC targets and T cell effectors from a single healthy donor (Fig. 5B). The CD4-based CARs suppressed infection by all HIV-1 isolates tested, with relative potencies similar to those described above (i.e., CD4-10-17b ≥ CD4 ≥ CD4-35-17b); again, the 139 control CAR did not suppress infection. The extreme potency of the CD4-10-17b CAR against all isolates (5- to 100-fold at a 0.2:1 ratio; 100- to 10,000-fold at a 1:1 ratio) is particularly notable.

Accessibility of 17b epitope upon Env engagement of CD4-based CARs.

We predicted that binding of HIV-1 pseudovirus particles to cells expressing the CD4 CAR should induce exposure of the 17b epitope, which would be detectable by flow cytometry using the 17b MAb. In contrast, with cells expressing the CD4-35-17b CAR, binding of the CD4 moiety to a gp120 subunit should induce exposure of the 17b epitope, enabling the 17b scFv moiety of the same CAR molecule to bind; thus, the 17b epitope would not be accessible to exogenously added 17b MAb. With the CD4-10-17b CAR, the linker is too short to enable simultaneous binding of the CD4 and 17b moieties to the same gp120 subunit; therefore, binding of the CD4 moiety should expose the 17b epitope to exogenously added 17b MAb, unless the 17b moiety from another CAR molecule were to occupy the epitope. We analyzed this issue by measuring exposure of the 17b epitope on HIV-1 pseudovirus particles captured by CD4-based CARs expressed on the surface of HOS cells. Flow cytometry analyses (Fig. 6, upper panels) clearly demonstrated the capture of HIV pseudovirions by cells expressing the various CD4-based CARs, as revealed with MAb 2G12 directed against a glycan epitope on the gp120 outer domain; bound pseudovirions were not detected on control cells (untransduced cells or cells expressing the negative-control DDY3 CAR). This result indicates that pseudovirion capture is mediated by specific binding of pseudovirion Env to the CD4 moiety of the various CARs. A very different pattern was observed with MAb 17b, directed against the CD4-induced epitope on gp120 (Fig. 6, lower panels). As expected, the epitope was readily detected on cells expressing the CD4 CAR but minimally on cells expressing the CD4-35-17b CAR, consistent with the expected bispecific binding of the 17b moiety following epitope exposure induced by the CD4 moiety; this interpretation was supported by the prominent binding of the 17b MAb to pseudovirions captured by the CD4-35-DDY3 CAR. Interestingly, minimal binding of 17b MAb was also observed with the CD4-10-17b CAR, despite the inability of the short linker to enable simultaneous binding of the CD4 and 17b moieties of a single CAR molecule to the same gp120 subunit; we interpret this result to reflect binding of the 17b moiety of a different CAR molecule to the CD4-induced epitope.

This research was funded in part by the Division of Intramural Research of NIAID and NCI and by the NIH Intramural AIDS Targeted Antiviral Program.

We thank Alon Hirschhorn and Joseph Sodroski for donating plasmid pGL4.22-JRFL and Dimiter Dimitrov for providing the DDY scFv cDNA. HIV-1 primary isolates were obtained from the NIH AIDS Reagent Program. Jack Bennink, Ron Germain, and Lawrence Samelson provided insightful discussions.