Journal: Biomolecules & Therapeutics

Article Title: Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy

doi: 10.4062/biomolther.2022.015

Figure Lengend Snippet: 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 (1×10 4 /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.

Article Snippet: Variable domains of the anti-CD3 (145-2C11) antibody ( Alegre et al ., 1995 ) were codon-optimized and synthesized commercially (Macrogen, Seoul, Korea).

Techniques: Generated, Cell Culture, Concentration Assay, Staining, Negative Control, Isolation, Incubation, Magnetic Beads, Activity Assay

Journal: Biomolecules & Therapeutics

Article Title: Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy

doi: 10.4062/biomolther.2022.015

Figure Lengend Snippet: 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 (2×10 6 /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 (2×10 5 /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.

Article Snippet: Variable domains of the anti-CD3 (145-2C11) antibody ( Alegre et al ., 1995 ) were codon-optimized and synthesized commercially (Macrogen, Seoul, Korea).

Techniques: In Vivo, Injection, Isolation, Magnetic Beads, Incubation, Activity Assay

Journal: Biomolecules & Therapeutics

Article Title: Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy

doi: 10.4062/biomolther.2022.015

Figure Lengend Snippet: 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 (2×10 6 /mouse) in their left flank and monitored for 30 days. BiTE (2 μg/kg), autologous polyclonal T cells, or BiTE:T cells (2×10 5 /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.

Article Snippet: Variable domains of the anti-CD3 (145-2C11) antibody ( Alegre et al ., 1995 ) were codon-optimized and synthesized commercially (Macrogen, Seoul, Korea).

Techniques: Construct, In Vivo, Injection, Activation Assay, Cell Culture