Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2–CH3 IgG spacer domains

Primary peripheral blood mononuclear cells (PBMCs) were isolated through leukapheresis of voluntary healthy donors by Ficoll density gradient centrifugation and stored in liquid nitrogen. Negative selection for isolation of CD3^pos T cells was performed using the Dynabeads Untouched Human™ T cell Kit (Life Technologies). T cells were further activated with Human T-Activator™ CD3/CD28 Dynabeads (Life Technologies) according to the manufacturer’s instructions (25 µl beads were used per 1 × 10⁶ T cells) and cultured in a medium consisting of RPMI GlutaMAX™ (Life Technologies) supplemented with 10% FCS (fetal calf serum, Sigma-Aldrich), 100 U/ml penicillin and 100 µg/ml streptomycin (both Life Technologies), i.e., a medium mix called “R-10”, plus 200 U/ml IL-2 (Peprotech). CD3/28-activated T cells were split every second day and cultured at densities between 0.5–1 × 10⁶ cells/ml. Primary human foreskin fibroblasts (HFF) were isolated from the foreskins of circumcised donors by mechanic disruption followed by enzymatic digestion with 5 mg/ml Collagenase D, 25 U/ml Dispase, and 0.05% Trypsin/EDTA. HFF were cultured in an R-10 medium and used between passage 10 and 18. Written informed consent was obtained from every donor’s parents, and the study protocols were approved by the ethics committee (Friedrich-Alexander-Universität Erlangen-Nürnberg no. 2247, Medizinische Universität Wien no. 514/2011). The hybridoma cell line “gB 27-287” [33] and the cell line IIA1.64 [34] were cultured in the R-10 medium. Neutralizing antibodies for IFN-γ (clone NIB42, Biolegend) and TNF (Infliximab; Centocor Ortho Biotech), and the respective isotype controls MOPC-21 and an anti-CD20 antibody (Rituximab, MabThera^®; Roche) were added to HFF cultures as indicated.

The gB-CAR was constructed using a single-chain variable fragment (scFv) derived from the antibody 27–287, as previously described [13]. For construction of CARs with different IgG-Fc parts, several mutations were introduced, as previously described [26, 35], into the spacers containing the Fc domain of either IgG1 or IgG4, i.e., substitution of ELLG (pos. 116–120) → PVA and S134A in IgG1-Fc (Uniprot ID P01857), and substitution of EFLB (pos. 113–116) → PVA in IgG4-Fc (Uniprot ID P01861). All CAR constructs were generated using the Gibson Assembly Kit (New England Biolabs) according to the manufacturer’s protocol. The resulting constructs were further amplified by PCR and used as templates for in vitro transcription.

Generation of infectious HCMV supernatants was performed by infecting semi-confluent HFF with the respective HCMV strain [multiplicity of infection (MOI) 0.1]. After 4 h, the remaining supernatant was discarded and fresh medium was added after washing it three times with R-10 to remove viral particles. Infected cells were cultured up to 14 days to achieve a high concentration of viral particles in the supernatant, which was subsequently harvested by centrifugation (2000 rpm, 10 min, 4 °C) and stored at − 80 °C until further use. Determination of the virus titer was performed by the limiting dilution method according to Reed and Munch. Infection of HFF for experiments with the respective HCMV strain was performed by 4-h co-incubation of the cells with thawed virus supernatant at an MOI 0.3 and 5 as indicated. Afterwards, the remaining supernatant was discarded and 3 washing steps with the R-10 medium were performed to remove remaining viral particles. Fresh medium was added and cells were further cultured at 37 °C. The HCMV strain AD169 encoding green fluorescent protein (GFP) was kindly provided by M. Marschall (Universitätsklinikum Erlangen, Germany), and the GFP-encoding HCMV strain Towne was a gift from B. Plachter (Universitätsmedizin, Johannes Gutenberg Universität Mainz, Germany).

To detect CARs on T cells and gB on HFF, the cells were first pre-incubated with 100 µg/ml murine IgG1-κ (clone MOPC21, Sigma-Aldrich) (10 min at 4 °C) before addition of the antibodies in order to prevent unspecific antibody-binding. In contrast, to detect FcRs on HFF and IIA1.64 cells, the cells were either not blocked before antibody addition or blocked, where indicated, with 10% human serum of an HCMV-negative individual (10 min, 4 °C). CAR expression in T cells was detected by incubation with a biotinylated anti-human IgG mab (clone JDC-10, Southern Biotec), followed by 2 washing steps and further incubation with PE-conjugated streptavidin (eBioscience). HCMV-gB was detected by incubation with supernatant from the hydridoma cell line gB-27-287 (final dilution 1:1) [33], followed by 2 washing steps and further incubation with a PE-conjugated anti-mouse antibody (eBiosicience). For detection of FcRs on the surface of HFF and IIA.64 cells, 0.5 × 10⁵ cells were incubated (without or with serum blocking, as indicated) with a fusion protein of IgG1-Fc coupled to CTLA4, termed CTLA4-IgG (5 µg/ml final concentration) (Bristol-Meyers Squibb Pharmaceuticals). After 2 washing steps, PE-conjugated anti-CTLA4 antibody (clone 14D3, eBioscience) was added to the cells. 7 AAD (eBioscience) was routinely added just before flow cytometric analysis in order to discriminate between viable and dead cells. All antibodies were incubated for 30 min at 4 °C. The washing buffer consisted of PBS (Life Technologies) supplemented with 10% FCS and 0.02% sodium azide (Merck KGaA). Flow cytometry was performed using a BD LSR Fortessa (BD Biosciences), and data were analyzed with FlowJo software (FlowJo Llc.). Cell counting was performed with Accucheck counting beads (Life Technologies), whereby dead cells were excluded by staining with propidium iodide.

The mRNAs encoding the CARs were generated by in vitro transcription from either 1 µg of linearized plasmid DNA or 50–200 ng of purified PCR products. In vitro transcription was performed using the mMessage mMachine T7 Ultra Kit (Ambion) according to the manufacturer’s protocol. In vitro transcribed mRNAs were purified with an adapted protocol using the RNeasy Kit (Qiagen). Briefly, RLT buffer from the kit and 1% beta-mercaptoethanol were added followed by the addition of absolute ethanol. After loading the sample onto an RNeasy column, purification was performed according to the manufacturer’s instructions. For electroporation of the mRNAs, 5 × 10⁶ cells were washed once with RPMI medium (Life Technologies) and once with Optimem (Life Technologies) (450×g, 5 min) to remove the remaining FCS and phenol-red. Afterwards, cells were resuspended in 100 µl Optimem, transferred into 4 mm electroporation cuvettes (VWR International GmbH) and electroporated with 10 µg mRNA using the Gene Pulser (Biorad) square wave protocol (500 V, 5 ms). The cells were used for in vitro assays 18–20 h after mRNA electroporation.

Supernatants were generated by co-culturing effector and target cells at an effector to target ratio of 2:1 in flat-bottom plates for 4 h at 37 °C. Prior to addition of the effector cells, the target cells (IIA1.64 or HFF) were routinely blocked with human serum of an HCMV-negative individual (10% final concentration, 15 min at 4 °C). Blocking with human serum was only omitted where indicated. The supernatants were collected, centrifuged (1600 rpm, 7 min) to remove remaining cells and stored at − 20 °C. IFN-γ in the supernatants was quantified using the Human IFN gamma ELISA Ready-SET-Go!^® Kit (eBioscience), and TNF was quantified using the Human TNF-α ELISA development kit (Mabtech). The limit of detection for IFN-γ and TNF was 4 and 13 pg/ml, respectively.

We previously generated a gB-specific CAR and showed that this CAR triggers T cell activation in response to HCMV infected cells. Since this does not result in substantial lysis of the infected cells, we asked if the CAR T cells could still efficiently inhibit HCMV replication by secretion of cytokines.

As a first step, we harvested supernatants of co-cultures of infected and non-infected HFF with T cells expressing either a gB-specific CAR or a CAR with irrelevant specificity [carcinoembryonic antigen (CEA)-specific CAR]. CAR expression in the T cells is depicted in Fig. 1a, and HCMV-gB expression in HFF is shown in Fig. 1b. Figure 1c and d illustrate that only CAR T cells expressing the gB-CAR specifically respond to HCMV-infected HFF and secrete IFN-γ and lower amounts of TNF. The blocking capacity of these supernatants was then tested in a subsequent experiment, in which HFF were infected with recombinant HCMV (strain AD169) encoding GFP under an immediate early promoter. This allowed for quantification of the fraction of infected HFF (green cells) by flow cytometry starting from 1 day after infection. Infection dose was low (MOI 0.3) in order to warrant that only a small fraction of HFF was initially infected (7.9–18.2% GFP^pos HFF on day 1; Fig. 2). Until day 4 after infection almost all of the HFF became GFP^pos (59.5–93.7%) due to reinfection with the newly replicated virus starting from day 3 after infection. This viral spread until day 4 was significantly inhibited (11.8–69.5% GFP^pos HFF) if cell-free supernatants from gB-CAR T cells (donors A–D) co-cultured with infected HFF were added simultaneously with the viral supernatant. Supernatants from the control conditions (T cells minus/plus irrelevant CAR, or co-culture with non-infected HFF) had no significant effect (Fig. 2). Additional file 1: Figure S1A and B depict the kinetics of infectious virus production in HFF (cell associated versus released particles after infection with HCMV at two different doses). This experiment was the basis for designing the above blocking trial and showed that new infectious virus particles first appear on day 3. The majority of the virus present on day 3 is cell associated. Release of free virus particles is low on day 3, but increases 100–1000-fold until day 5, whereas cell-associated particles increase only slightly.

In the next step we wanted to investigate which factors secreted by the gB-CAR T cells inhibit the replication of HCMV. Early studies showed that type I interferons but also the T cell secreted cytokines IFN-γ and TNF can have a strong antiviral capacity [24, 25]. So, we repeated the infection experiment explained above with supernatants from gB-CAR T cells plus/minus neutralizing antibodies against IFN-γ and TNF. Figure 3 illustrates that the inhibitory effect of the co-culture supernatants (T cell donors A-D) on HCMV replication in HFF until day 4 was almost completely abrogated by the combined neutralization of IFN-γ and TNF. The neutralization of either IFN-γ or TNF alone had a smaller effect, and, as expected, the addition of the isotype control antibodies had no effect on the inhibition of HCMV replication by the supernatants.

Overall these data demonstrate that the gB-CAR mediates inhibition of HCMV replication independent of cytotoxic effector functions via the combined action of IFN-γ and TNF, which are released by the activated CAR T cells.

Our original gB-specific CAR construct contained a CH2–CH3 Fc domain that was meanwhile recognized to abrogate CAR T cell function in vivo [26‐30]. However, since HCMV encodes at least four different IgG-Fc binding proteins in order to escape from antibody-mediated immune responses [31], we speculated that this CH2–CH3 domain after mutation could be particularly attractive for targeting HCMV.

As a first step we analyzed the kinetics of HCMV-FcR expression by determining the capacity of infected HFF for binding of human IgG1-Fc. Figure 4a illustrates that IgG1-Fc could strongly bind to HFF starting from day 3 after infection with HCMV strains AD169 and Towne. For these experiments we used IgG1-Fc fused to CTLA4, which was detected by second-step staining with an anti-CTLA4 antibody. Binding of the CTLA4-Fc to infected HFF was completely blocked by pre-incubation of the cells with human serum, demonstrating that the interaction is mediated by the Fc domain (Fig. 4b). The same procedure plus/minus prior serum blocking was applied for detection of human CD64 in the murine cell line IIA1.64 [34] which expressed high levels of CD64 (Fig. 4c) and was used for control. In a next step we co-cultured CAR T cells with infected or non-infected HFF or IIA1.64 cells and determined the secretion of IFN-γ (Fig. 5). IFN-γ levels were normalized to the levels obtained with the CD64^high control cell line IIA1.64. A CEA-specific CAR was included as a non-relevant CAR that could only be activated by the infected HFF via the integrated CH2–CH3 spacer. Figure 5 shows that both the gB-specific and the CEA-specific CAR, in the absence of serum blocking, efficiently recognized the CD64^high IIA1.64 cells and the infected HFF, whereas there was no specific response towards uninfected HFF. As expected, pre-incubation of the target cells with human serum completely blocked IFN-γ production in T cells, when the interaction with target cells was only mediated via the CH2–CH3 domain in the CAR. This was the case when the CAR T cells were co-cultured with IIA1.64 cells or when CEA-specific CAR T cells were co-cultured with infected HFF. However, when the T cells were directed against infected HFF via the gB-specific CAR, which could additionally interact via the gB-specific scFv, then IFN-γ secretion was only partially blocked by the human serum. Notably, the same T cells were completely blocked by human serum in co-culture with IIA1.64 cells.

Our data confirm two points: First, that HCMV-encoded FcRs similarly to gB occur on the surface of infected cells starting from day 3 after infection and hence could serve as target antigens. Second, the data show that the HCMV-FcRs indeed interact with and efficiently trigger activation in T cells expressing CARs with integrated CH2–CH3 domains.

Since the recognition of human endogenous FcRs by the CH2–CH3 domain results in reduced efficacy of the CAR T cells in vivo, a wild-type CH2–CH3 domain for recognition of HCMV-FcRs is not an option. However, there is evidence that the mode of interaction of HCMV-FcRs could substantially differ from that of endogenous FcRs [32]. We hypothesized that introducing specific mutations inhibiting the interaction of the CH2–CH3 domain with endogenous FcRs (except the neonatal FcR) could still allow for interaction with HCMV-FcRs.

Consequently, we generated CARs with mutated CH2–CH3 domains of IgG1 and IgG4, which were previously reported to not interact with endogenous FcRs and to rescue CAR function in vivo [26, 35]. These CH2–CH3 domains (Fig. 6a) were integrated into a non-gB CAR, which was directed against CD19, and not CEA, since it turned out that mutated CH2–CH3 domains further reduced the expression of the CEA-specific CAR, which already showed reduced expression on its own. This unwanted reduction was not observed with the CD19-specific CAR (Fig. 6b). The CD19-specific CARs with either the mutated Fc domain of IgG1 or of IgG4 indeed mediated the recognition of HCMV-infected HFF, which again was inhibited by pre-incubation of the HFF with human serum (Fig. 7a). The cytokine levels achieved with CARs containing the mutated Fc domains, however, were reduced compared to the CAR with a wild-type CH2–CH3 (Fig. 7a). Notably, also the antigen independent background secretion of IFN-γ in the co-cultures with non-infected HFF was reduced in T cells with mutated CH2–CH3 domains (Fig. 7b), possibly indicating a generally reduced capacity for cytokine secretion in the cells resulting from expression of an unstable mutated protein domain.

These experiments prove that CARs with mutated IgG1- and IgG4-Fc domains indeed allow for recognition of HCMV-infected cells without targeting endogenous FcRs.