Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy is one of the promising anticancer treatments. It shows a high overall response rate with complete response to blood cancer. However, there is a limitation to solid tumor treatment. Additionally, this currently approved therapy exhibits side effects such as cytokine release syndrome and neurotoxicity. Alternatively, bispecific antibody is an innovative therapeutic tool that simultaneously engages specific immune cells to disease-related target cells. Since programmed death ligand 1 (PD-L1) is an immune checkpoint molecule highly expressed in some cancer cells, in the current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) which can engage T cells to PD-L1⁺ cancer cells. We observed that the BiTE-bound OT-1 T cells effectively killed cancer cells in vitro and in vivo. They substantially increased the recruitment of effector memory CD8⁺ T cells having CD8⁺CD44⁺CD62L^low phenotype in tumor. Interestingly, we also observed that BiTE-bound polyclonal T cells showed highly efficacious tumor killing activity in vivo in comparison with the direct intravenous treatment of bispecific antibody, suggesting that PD-L1-directed migration and engagement of activated T cells might increase cancer cell killing. Additionally, BiTE-bound CAR-T cells which targets human Her-2/neu exhibited enhanced killing effect on Her-2-expressing cancer cells in vivo, suggesting that this could be a novel therapeutic regimen. Collectively, our results suggested that engaging activated T cells with cancer cells using αCD3xαPD-L1 BiTE could be an innovative next generation anticancer therapy which exerts simultaneous inhibitory functions on PD-L1 as well as increasing the infiltration of activated T cells having effector memory phenotype in tumor site.

Keywords: Anticancer immunotherapy; Bispecific T cell engager; Bispecific antibody; CAR-T; PD-L1.

Fig. 1

Generation of bispecific T-cell engager-bound T cells (BiTE:T cells) as a novel anticancer cell therapy. (A) Bispecific T-cell engager molecules were generated with two single chain fragment variables (scFvs). mCD3xPD-L1 BiTE contains scFvs of anti-CD3ε antibody (145-2C11) and anti-PD-L1 antibody (KL001-13), which were linked with ‘GGGGS’ linker. (B) mCD3xPD-L1 BiTE was cultured with CD8⁺ T cells for 1 h or 48 h in following concentration: 0.01 μg/mL, 0.1 μg/mL, and 1 μg/mL. T cells were stained with 1 μM of CellTrace^TM Violet (CTV) and dilution of CTV was measured with FACS to determine T cell proliferation. Non-treated T cells were used as a negative control and indicated it as red line in figure. ***p<0.001, ****p<0.0001 (Student’s t-test; n=3/group). (C) Schematic diagram of BiTE-bound T cells (BiTE:T cells) preparation. To prepare BiTE:T cells, CD8⁺ T cells were isolated from the spleen of mice and incubated for 48 h with 4 μg/mL of αCD3 antibody and 2 μg/mL of αCD28 antibody. Then mCD3xPD-L1 BiTE (0.1 μg/mL) were incubated with the CD8⁺ T cells for 1 h. BiTE-bound CD8⁺ T cells (BiTE:T cells) were obtained after removing the unbound BiTEs by washing with PBS. (D) CD8⁺ T cells from wild type mice isolated using magnetic beads. T cells or BiTE:T cells were prepared following protocol shown in 1C. T cells or BiTE:T cells were incubated with MO5 cells (1×10⁴/well) for 16 h. Represented ratio is Target cell:Effector cell. Cancer killing activity of BiTE:T cells or BiTE:OT-1 cells was measured by CCK8 and CTV assay.

Fig. 2

In vivo antitumor effect of BiTE:OT-1 cells. (A) Scheme of the in vivo experiment for BiTE:OT-1 T cells. Groups of mice were subcutaneously injected with MO5 (2×10⁶/mouse) in their left flank and the tumor size was monitored for 28 days. C57BL/6 mice, which were transplanted with MO5, were injected with OT-1 T cells or BiTE:OT-1 T cells (2×10⁵/mouse) after 10 and 20 days of tumor cell injection. (B) Tumor size was measured in every 2 or 3 days. All groups of mice were sacrificed on day 28. *p<0.05, **p<0.01 (Student’s t-test; n=6/group). (C) CD8⁺ T cells from wild type mice or OVA-specific OT-1 CD8⁺ T cells (>90% of which were Vα2-positive) isolated from OT-1 mice using magnetic beads. BiTE:T cells or BiTE:OT-1 cells were prepared following protocol shown in 1C. BiTE:T cells or BiTE:OT-1 cells were incubated with MO5 cells for 16 h. Cancer killing activity of BiTE:T cells or BiTE:OT-1 cells were measured by CCK8 and CTV assay. ***p<0.001 (Student’s t-test; n=3/group). (D) Tumor-infiltrating lymphocytes (TILs) were analyzed. Effector memory T cells (CD3⁺CD8⁺ CD62L^low) were analyzed by FACS. The population and number of them were shown. **p<0.01, ***p<0.001 (Student’s t-test; n=4/group). These data are representative of three independent experiments.

Fig. 3

Antitumor effect of BiTE:T cells constructed with activated autologous polyclonal CD8⁺ T cells. (A) Scheme of the in vivo experiment for BiTE-bound autologous polyclonal T cells (BiTE:T cells). Groups of mice were subcutaneously injected with MO5 (2×10⁶/mouse) in their left flank and monitored for 30 days. BiTE (2 μg/kg), autologous polyclonal T cells, or BiTE:T cells (2×10⁵/mouse) were intravenously transferred to tumor-bearing mice after 10 days and 20 days of tumor injection. (B) Tumor sizes were monitored for every 2 days and the tumor weights were measured at day 30 after euthanizing the mice. **p<0.01, ***p<0.001 (Student’s t-test; n=6/group). (C) CD3⁺CD8⁺CD44⁺CD62L⁺ effector memory T cells and CD3⁺CD8⁺CD44⁺CD62L^– central memory T cells in tumor infiltrating lymphocytes (TILs) of every group of mice were analyzed by FACS. *p<0.05, **p<0.01 (Student’s t-test; n=4/group). (D) MO5 cells were grafted in the left flank of C57BL/6 and BiTE (2 μg/kg), and BiTE-bound T cells (BiTE:T) with or without activation through αCD3 and αCD28 were intravenously injected after 10 days and 20 days of tumor challenge. Tumor sizes were monitored for every 2 days or 3 days. Non-activated T cells were also cultured for the same period as the activated T cells. After euthanizing the mice, tumor weight was measured. *p<0.05, **p<0.01 (Student’s t-test; n=4/group). All the data shown are representative of three independent experiments.

Fig. 4

Novel antitumor cell therapy of BiTE:ML39 CAR-T. (A) Diagram of the in vivo study for BiTE-bound ML39 CAR-T cells (BiTE:ML39 CAR-T). Groups of mice were grafted with Her-2/CT26 cells (2×10⁶/mouse) into their left flank and monitored for 30 days. ML39 CAR-T cells were generated to express anti-hHer2/neu (ErbB2) for targeting the Her-2/CT26 cells. After 10 and 20 days of tumor injection, mice were injected with control CAR-T cells (CTL CAR-T), ML39 CAR-T cells, or BiTE:ML39 CAR-T cells (2×10⁵/mouse). (B) Confirmation of PD-L1 expression on the surface of Her-2/CT26 cells. (C) Tumor sizes were monitored for every 2 days. *p<0.05, ***p<0.001 (Student’s t-test; n=10/group). (D) After euthanizing every group of mice, the tumor was removed from their body and the tumor mass was measured. *p<0.05, ***p<0.001 (Student’s t-test; n=5-8/group). These data are representative of three independent experiments.