Figure 5.

Treatment with the SAR T cell-BiAb combination is effective in syngeneic and xenograft melanoma models and enhances survival in vivo. (A) C57BL/6 mice were injected i.p. weekly with an anti-CD20 depleting antibody (250 µg/injection) starting 7 days before tumor cell injection. Mice were inoculated intravenously with 2×10⁶ YUMM1.1 TYRP1-LUC-GFP tumor cells. Mice were treated with a single intravenous injection of T cells 4 days after tumor cell injection. Simultaneously, antibody treatment was given by i.p. injections of the αTYRP1/αE3 BiAb (αTYRP1, 5 µg/injection) which was redosed two times per week. In vivo luminescent signal imaging was performed one time per week using IVIS. Treatment groups were as follows: SAR T cells and αTYRP1/αE3 BiAb (n=10), E3del T cells and αTYRP1/αE3 BiAb (n=5), SAR T cells (n=5), Unt T cells and αTYRP1/αE3 BiAb (n=4), and the vehicle solution (n=9). (B) Percentage survival readout. (C) Tumor burden per mg of lung tissue 19 days after T cell therapy using a flow cytometry-based readout. Mice were treated with either SAR T cells and αTYRP1/αE3 BiAb (n=5), Unt T cells and αTYRP1/αE3 BiAb (n=4) or SAR T cells only (n=5) 4 days after tumor induction. (D) Immunofluorescence imaging of the αTYRP1/αE3 BiAb and tumor cell-derived GFP in lung tissue was carried out with anti-human IgG and anti-GFP stainings. Mice were injected intravenous either with 2×10⁶ YUMM1.1 TYRP1-LUC-GFP cells or with vehicle solution. After 20 days the mice were injected either with 5 µg αTYRP1/αE3 BiAb, an isotype BiAb or with the vehicle solution. Following 48 hours of incubation, the lung, heart and skin were harvested, stained and imaged using ZellScannerONE. The groups were as follows: Tumor and αTYRP1/αE3 BiAb (n=3), αTYRP1/αE3 BiAb only (n=2), tumor and isotype BiAb (n=3). (E) NSG mice were inoculated s.c. with 1×10⁶ MV3 tumor cells. Mice were treated with a single intravenous injection of T cells 5 days after tumor cell injection. Simultaneously, antibody treatment was given by i.p. injections of the αMCSP/αE3 BiAb (αMCSP, 5 µg/injection) which was redosed two times per week. Treatment groups were as follows: SAR T cells and the αMCSP/αE3 BiAb (n=5), Unt T cells and αMCSP/αE3 BiAb (n=4), SAR T cells only (n=5), BiAb only (n=5), and the vehicle solution (n=5). (F) In an endpoint experiment, SAR T cell persistence in the blood and in the tumor was analyzed using a flow cytometry-based readout 14 days after T cell transfer. The mice were treated with either SAR T cells and αMCSP/αE3 BiAb, Unt T cells and αMCSP/αE3 BiAb or SAR T cells only (for each group n=5). (G) SAR T cell infiltration per mm³ tumor. (H) Frequency of PD-1 expression on human CD4⁺ and CD8⁺ T cells in the tumor. (I) NSG mice were injected s.c. with 0.2×10⁶ A375 tumor cells. The mice were treated according to the experiment in (E) 11 days after tumor induction (for E3del and αMCSP/αE3 BiAb: n=4, for other groups: n=5). (J) Percentage survival readout. (K) NSG mice were injected s.c. with 0.4×10⁶ patient-derived melanoma cells (patient sample 2). The mice were treated according to the experiment in (E) 12 days after tumor induction with αTYRP1/αE3 BiAb (for E3del and αTYRP1/αE3 BiAb, and vehicle solution: n=4, for other groups: n=5). For statistical analysis of survival data, the log-rank test was applied. Analyses of differences between groups for (E), (I) and (K) were performed using two-way analysis of variance with correction for multiple testing by the Bonferroni method. For statistical analysis of (C), (F), (G) and (H) the unpaired two-tailed Student’s t-test was used. Experiments show mean values±SEM calculated from n biological replicates, one experiment for (B), (C), (F), (G), (H) and (K), and one representative of two independent experiments in (A), (E), (I) and (J). BiAb, bispecific antibodies; i.p., intraperitoneally; MCSP, melanoma-associated chondroitin sulfate proteoglycan; PD-1, programmed cell death protein 1; PDX, patient-derived xenograft; SAR, synthetic agonistic receptor; s.c., subcutaneously; TYRP1, tyrosinase-related protein 1; Unt, untransduced T cells.