Bispecific Antibodies Enable Synthetic Agonistic Receptor-Transduced T Cells for Tumor Immunotherapy

Abstract

Purpose: Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing.

Experimental design: BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell-induced lysis were characterized in three murine and human tumor models in vitro and in vivo.

Results: Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo.

Conclusions: We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.

Figure 1. Synthetic agonistic receptor (SAR) T cells can be triggered by BiAb to induce T cell activation, proliferation and tumor celllysis.

(A) Schematic overview of the constructs used in experiments for panels B to H: tetravalent 2 + 2 anti-EpCAM x anti-EGFRvIII (or anti-Cripto) BiAb and receptor constructs E3, E3^del, C3 and C3^del (B) Quantification of E3, E3^del, C3, C3^del and untransduced murine T cell activation when cultured in 96-well plates, coated with the corresponding 2 + 2 BiAb (10 μg/ml). (C, D, G, H) Transduced T cells were co-cultured with Panc02-OVA-EpCAM⁺ tumor cells and 2 + 2 BiAb (1 μg/ml). The absolute number and the proliferation rate of the T cells was determined by staining cells for intracellular Ki67 before and after co-culture. For phenotypic analysis, T cells were stained for CD8, CCR7 and CD62L surface expression. CD8⁺CCR7⁺CD62L⁺ T cells were defined as central memory T cells and CD8⁺CCR7^-CD62L^- T cells as effector memory T cells. (E) Quantification of B16-EpCAM tumor cell lysis when cocultured with E3, E3^del, C3 or C3^del T cells (E:T 10:1). (F) Real-time lysis of 4T1 tumor cells when cultured with E3 T cells with or without 1 μg/ml of BiAb (E:T 10:1). All graphs show mean values ± SEM of at least triplicates. Experiments in panels C, D, G and H were performed in duplicates for technical reasons. Each experiment shown is representative of at least three independent experiments. p-values by two-sided unpaired t-test are indicated. For impedance-based cytotoxicity assays the total curve over time was compared. p<0.05 was considered statistically significant.

Figure 2. Antibodies monovalent (vs. bivalent) for binding SAR avoid tumor-antigen independent T cell activation.

(A) Schematic overview of the constructs used in panels B to G: trivalent (2+1 format) and a tetravalent (2 + 2 format) anti-EpCAM x anti-EGFRvIII BiAb as well as the constructs E3 and E3^del. (B) Quantitative analysis of E3, E3^del or untransduced T cell activation when cultured with coated or soluble human EGF or different anti-EGFR antibodies (cetuximab or panitumumab, all at 10μg/ml) for 48 h. (C) Culture of E3 T with either immobilized or soluble 2 + 2 BiAb in increasing concentrations (0, 0.1, 1 or 10 μg/ml). (D) Quantification of E3 T cell activation when cultured with either coated or soluble 2 + 2 BiAb or 2 + 1 BiAb in increasing concentrations (0, 0.1, 1 or 10 μg/ml). (E, F) Co-culture of Panc02-OVA (E) or Panc02-OVA-EpCAM⁺ (F) tumor cells with E3 T cells (E:T 5:1) preloaded with decreasing amounts (1, 0.5, 0.25, 0.125 or 0.062 μg/ml) of either 2 + 2 BiAb or 2 + 1 BiAb. (G) Saturation curve analysis of E3 T cells for 2 + 2 BiAb and 2 + 1 BiAb BiAb in relation to the lysis curves of Panc02-OVA-EpCAM⁺ tumor cells. All graphs show mean values ± SEM of at least triplicates. Cytotoxicity analysis E and F were performed in quadruplicates. Experiments shown are representative of at least three independent experiments. p-values by two-sided unpaired t-test are indicated. p<0.05 was considered statistically significant.

Figure 3. Tumor cell killing by SAR T cells relies on Fas-FasL interactions and is independent of granzyme and perforin release.

(A) Schematic overview of the constructs used in panels B to E: the trivalent antibody and the constructs E3. (B) Real-time Panc02-OVA-EpCAM⁺ tumor cell killing with either E3 wild-type or granzyme B knock-out murine T cell (E:T 10:1). (C) Quantification of Panc02-OVA-EpCAM⁺ tumor cell lysis mediated by either E3 wild-type or perforin knock-out murine T cells (E:T 10:1). (D) Impact of T cell-induced tumor cell apoptosis blocking antibodies (anti-CD178 [anti-FasL] or anti CD253 [anti-TRAIL]; each at 10 μg/ml) on Panc02-OVA-EpCAM⁺ tumor cell lysis by E3 T cells (E:T 10:1). (E) Real-time Panc02-OVA-EpCAM⁺ tumor cell killing by either E3 wild-type or Fas ligand knock-out murine T cells (E:T 10:1). All graphs show mean values ± SEM of at least triplicates. Impedance-based cytotoxicity assays were performed in duplicates for technical reasons. Experiments shown are representative of at least three independent experiments. p-values by two-sided unpaired t-test are indicated. For impedance-based cytotoxicity assays the total curve over time was compared. p<0.05 was considered as statistically significant.

Figure 4. Anti-human mesothelin x anti-EGFRvIII 2 + 1 BiAb specifically stimulates SAR T cells and mediates tumor killing only in the presence of mesothelin-positive cells.

(A) Schematic overview of the constructs used in experiments for panels B to E: Trivalent BiAb as well as the constructs E3 and E3^del. (B) E3, E3^del or untransduced T cell activation when cultured with either coated or soluble 2 + 1 BiAb (5 μg/ml). (C) Quantification of E3, E3^del and untransduced T cell activation in co-culture with Suit-2-MSLN tumor cell (E:T 10:1). (D) Real-time Suit-2-MSLN tumor cell lysis by E3 or E3^del T cells with indicated effector to target ratio. (E) NSG mice inoculated s.c. with Suit-2-MSLN were treated with a single i.v. injection of E3 T cells only (n=5), E3 T cells + 20 μg BiAb (n=10), E3 T cells + 5 μg BiAb (n=5), E3^del T cells + 20 μg BiAb (n=5), C3 T cells + 20 μg BiAb (n=5), 20 μg BiAb only (n=5), and PBS (n=5), followed by a total of four BiAb injections as indicated. (F) NSG mice inoculated s.c. with MIA PaCa-MSLN were treated with a single i.v. injection of E3 T cells only (n=6), E3 T cells + 10 μg BiAb (n=10), E3^del T cells + 10 μg BiAb (n=6), C3 T cells + 10 μg BiAb (n=6), 10 μg BiAb only (n=6), and PBS (n=6), followed by a total of ten BiAb injections as indicated. (G) NSG mice inoculated s. c. with MSTO-MSLN-LUC were treated with a single i. v. injection of E3 T cells only (n = 5), E3 T cells + 10 μg BiAb (n = 5), 10 μg BiAb only (n = 5) and PBS (n = 5), followed by a total of four BiAb injections as indicated. (H, J) Tumors from the mice in experiment 4G were FACS-analyzed for T cell infiltration. Following a drill down through the CD45 and CD3 gates, the EGFR+ CD4 (H) and CD8 (J) T cell populations were quantified and are depicted as a ratio of cells per bead. In vitro experiments show mean values ± SEM of at least triplicates and are representative of at least three independent experiments. p-values are based on two-sided unpaired t-test. Impedance-based cytotoxicity assays were performed in duplicates for technical reasons. For impedance-based cytotoxicity assays the total curve over time was compared. In vivo experiments shown are representative of two independent experiments. Analysis of differences between groups for the tumor growth curves were performed using two-way ANOVA with correction for multiple testing by the Bonferroni method. p<0.05 was considered as statistically significant.

Figure 5. E3-MSLN BiAb selectively activates E3 T cells while its depletion results in rapid and complete reversibility of SAR T cell stimulation.

(A, B) For BiAb depletion, Suit-2-MSLN (A) or Panc02-OVA-EpCAM (B) tumor cells were repeatedly co-cultured with E3 T cells with or without readjustment of the BiAb concentration (5 μg/ml). Control condition utilized anti-MSLN-CAR or anti-EpCAM-CAR T cells respectively (E:T 10:1). (C) Analysis of remaining E3-GFP T cells in spleen, lymph node, blood and lung after cetuximab administration. (D, E) NSG mice inoculated s.c. with Suit-2-MSLN (n = 5 / group) were treated with a single i.v. injection of E3 T cells only, E3 T cells + 10 μg BiAb - redosed, E3 T cells + 10 μg BiAb - single dose and PBS, followed by a total of six BiAb injections as indicated. * indicates significance of non-redosed group vs. E3 only at day 12 (p = 0.026) (E). Mice were bled 3, 6, 10 and 16 days after T cell transfer to quantify T cell activation by IFN-γ serum levels (F). Data are reported normalized to the values obtained from the single dose group. In vitro and in vivo experiments show mean values ± SEM and are representative of at least two independent experiments. p-values are based on two-sided unpaired t-test. Analysis of differences between groups for the tumor growth curves were performed using two-way ANOVA with correction for multiple testing by the Bonferroni method. p<0.05 was considered as statistically significant.