TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen sensitivity combined with high fidelity of activation

Abstract

Low antigen sensitivity and a gradual loss of effector functions limit the clinical applicability of chimeric antigen receptor (CAR)-modified T cells and call for alternative antigen receptor designs for effective T cell-based cancer immunotherapy. Here, we applied advanced microscopy to demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform second-generation CAR formats with regard to conveyed antigen sensitivities by up to a thousandfold. TCC-based antigen recognition occurred without adverse nonspecific signaling, which is typically observed in CAR-T cells, and did not depend-unlike sensitized peptide/MHC detection by conventional T cells-on CD4 or CD8 coreceptor engagement. TCC-endowed signaling properties may prove critical when targeting antigens in low abundance and aiming for a durable anticancer response.

Fig. 1.. Protein-functionalized SLB-based platform for high-throughput analysis of TCR and synthetic antigen receptor–mediated activation and downstream signaling events.

(A) Schematic representation of an SLB equipped with fluorescently labeled antigens A2/CMV or CD19, ICAM-1 adhesion molecules for recognition by RA14 TCR T cells or CD19 BBz CAR T cells. Extracellular portions of proteins are extended with a polyhistidine tag (12× His) to interact with 18:1 DGS-Ni-NTA (schematically depicted as red circles) present in the SLB. (B) A total of 12.5% reducing SDS–polyacrylamide gel electrophoresis followed by silver staining of the recombinant proteins used for SLB functionalization. (C) FRAP was used to determine the mobile fraction of SLB-anchored A2/CMV and CD19. Fluorescence intensities were normalized to the initial intensity values and plotted over time. Data shown are representative of n = 3 biological replicates. (D) Micrographs of A2/CMV-AF647–decorated SLBs with densities indicated (white). Scale bar, 5 μm. (E) Micrographs showing recruitment of A2/CMV-AF647 (by RA14 TCR T cells) and CD19-AF647 (by CD19 BBz CAR T cells) on SLBs featuring ICAM-1 in addition to the respective antigen. Antigen densities indicated (white). Scale bar, 5 μm. (F) Antigen-dependent changes in intracellular calcium concentrations as monitored by Fura-2 time-lapse microscopy in a single CD19 BBz CAR T and a single RA14 TCR T cell confronted with SLBs functionalized with ICAM-1 as well as CD19 or A2/CMV in indicated densities. Scale bar, 10 μm. (G) Normalized Fura-2 ratio values of T cells shown on the left as a function of time. DIC, differential interference contrast.

Fig. 2.. Calcium and downstream signaling response of T cells equipped with CMV-specific RA14 TCR and anti-CD19 second-generation CAR T cells.

(A) Histograms of calcium response of RA14 TCR T cells facing A2/CMV (left), RA14 TCR T cells confronted with A2^ΔCD8/CMV (middle), and CD19 BBz CAR T cells exposed to CD19 (right) at indicated densities. Dashed lines indicate Fura-2 ratio thresholds above which T cells were considered activated. (B) Top: Calcium response of RA14 TCR T cells and CD19 CAR T cells shown in (A) plotted as dose-response curves. Data were fitted to Eq. 1 as summarized in Table 1. Middle: Mean Fura-2 ratio values [taken from (A)] measured for the activated fraction of CD19 BBz CAR- and RA14 T cells confronted with SLBs featuring CD19, A2/CMV, or A2^ΔCD8/CMV at indicated densities. Data were pooled from n = 2 biological replicates with n = 2 donors. Bottom: Quantification of IFN-γ secreted into the media after 72-hour stimulation of RA14 TCR and CD19 BBz CAR T cells with SLB-resident A2/CMV and CD19, respectively. Data are representative of one donor. Statistics: Mean ± SEM of technical duplicates.

Fig. 3.. Design and surface expression of synthetic antigen receptor constructs.

(A) Schematic representation of RA14 TCR, 1G4 TCR, affinity-enhanced c58c61 1G4 TCR (1G4hi TCR), synthetic receptors T1 STAR_nat and T1 STAR_mir (based on the murine TCRαβ), T1 or FMC63 scF_V tethered to CD3ε via a flexible linker (εTRuC), T1-scF_V (T1) BBz CAR, and FMC63 scF_V (CD19) BBz CAR. (B) Left: Histograms depicting flow cytometric surface analysis of CRISPR-Cas9–engineered (solid line) and lentivirally transduced (dashed line) antigen receptor modified T cells as performed with indicated probes (FACS plots, left) to determine the mean number of surface-expressed antigen receptors (dot plot, right). Right: Individual flow cytometric measurements are indicated for receptor modifications involving CRISPR-Cas9–based knock-in (filled circles) and lentiviral transduction (empty circles) of T cells derived from up to four donors. Quantitation of surface receptors was based on label saturation and flow cytometric calibration (fig. S2, C and D). (C and D) TIRF images of living T cells modified with indicated antigen receptors, stained as indicated with probes under saturating conditions, confronted with ICAM-1–functionalized SLBs and quantitated for respective surface densities. Data represent n = 21 to 47 synapses of T cells derived from one donor. Scale bar, 5 μm. Statistics: Mean ± SD.

Fig. 4.. Calcium response of T cells equipped with NY-ESO-1–specific TCR, TCC, or CARs targeting peptide-loaded HLA.A2.

T cells modified with a knocked-in 1G4 TCR, 1G4hi TCR, T1 STAR_nat, T1 STAR_mir, T1 BBz CAR, or with a lentivirally transduced T1 εTRuC were confronted with SLBs functionalized with A2/9V, A2/6T, and A2/4D at indicated densities. Antigen dose-calcium response (left) and mean Fura-2 ratio values of activated T cells (right) are shown (refer to fig. S5 for histogram-based analysis). Data were fitted to a three-parameter dose-response curve according to Eq. 1 to extract EC₅₀ values and confidence intervals as summarized in Table 1. Data were pooled from n = 2 to 3 biological replicates of two to three donors.

Fig. 5.. Calcium response of T cells equipped with NY-ESO-1–specific TCR, TCC, or CARs targeting peptide-loaded CD8 binding–deficient HLA.A2^ΔCD8.

T cells modified with a knocked-in 1G4 TCR, 1G4hi TCR, T1 STAR_nat, T1 STAR_mir, T1 BBz CAR, or with a lentivirally transduced T1 εTRuC were confronted with SLBs functionalized with A2^ΔCD8/9V, A2^ΔCD8/6T, and A2^ΔCD8/4D at indicated densities. Antigen dose-calcium response (left) and mean Fura-2 ratio values of activated T cells (right) are shown (refer to fig. S5 for histogram-based analysis). Data were fitted to a three-parameter dose-response curve according to Eq. 1 to extract EC₅₀ values and confidence intervals as summarized in Table 1. Data were pooled from n = 2 to 3 biological replicates of two to three donors.

Fig. 6.. Comparative heatmap of T cell antigen sensitivities conveyed by antigen receptors.

Log fold increase in antigen threshold (EC₅₀ value) required for T cell activation (normalized to the activation threshold of A2/CMV-confronted RA14 T cells) for indicated receptor constructs and ligands. Numbers in smaller font indicate the bottom and top 95% confidence intervals of the EC₅₀ value normalized to the EC₅₀ value of the RA14 T cells. Antigen receptor–expressing T cells marked with an asterisk (*) were lentivirally transduced. All other constructs were introduced via CRISPR-Cas9. Nan, not a number; n.d., not determined.

Fig. 7.. Calcium response of T cells equipped with a CD19-reactive second-generation CAR or an εTRuC.

(A) Histograms depicting population-wide analysis of the calcium response of CD19 BBz CAR- and CD19 εTRuC T cells confronted with SLBs functionalized with CD19 at indicated densities as well as ICAM-1. Dashed lines indicate the Fura-2 ratio threshold above which T cells were considered activated. (B) Top: Rendering of data shown in (A) as antigen dose-response curves. Data were fitted to Eq. 1 as summarized in Table 1 (only εTRUC shown). Bottom: Mean Fura-2 ratio values measured for the activated fraction of CD19 BBz CAR- and CD19 εTRuC T cells [see (A)] confronted with SLBs featuring CD19 at indicated densities. Data were pooled from n = 3 biological replicates of three donors.

Fig. 8.. Ex vivo cytotoxic capacity of engineered T cells with low antigen receptor expression levels.

1G4 T cells were engineered via CRISPR-Cas9; all other constructs were lentivirally transduced [refer to Fig. 3 (B to D)]. Effector cells were cocultured for indicated times at a ratio of 1:1 and with HLA.A2-, CD80-, and luciferase-expressing K562 feeder cells, prepulsed at specified concentrations with the 9V NY-ESO-1 peptide derivative. Statistics: Mean ± SEM from technical duplicates of one donor.

Fig. 9.. Immunofluorescence-based analysis of synapse-associated CD3ζ pITAM#2 and pZAP70.

(A) Synaptic immunofluorescence recorded in TIRF mode with the use of indicated antibodies. Shown are synapses of T cells modified with indicated antigen receptors and in contact with SLBs featuring ICAM-1 and, if indicated, their nominal antigen (A2/CMV, A2/9V, and CD19, respectively). (B) Synapse-associated integrated fluorescence of CD3ζ Y111, 123 ITAM no. 2 in the absence (left) and presence of antigen (right) as indicated. Data are representative of n = 20 to 48 recorded T cell synapses per condition of one donor. Statistics: Mean ± SEM. (C) Synapse-associated integrated fluorescence of ZAP70 pY319 in the absence (left) and presence of antigen (right) as indicated. Data are representative of n = 21 to 60 recorded T cell synapses per condition of one donor. Statistics: Mean ± SEM. (D) Ratios of pZAP70 and pITAM#2 integrated fluorescence intensities [from (B) and (C)] which had been normalized to ratio values measured for CD19 BBz CAR T cells. For analysis, integrated pZAP70 fluorescence values had been extrapolated for antigen densities associated with measured integrated pITAM#2 fluorescence values.