Fig. 3.

The bi-specific scFv antibody (BsAb) delays tumor growth and modulates intratumoral cytokines and the proportions of infiltrating immune cells, MDSCs, and M2 TAMs. (A) Schematic representation of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 10⁶ MC38 cells expressing CEA. On day 7, mice received intravenous injection of PD-1 scFv, TREM2 scFv, or the BsAb at 200 µg/mouse daily; the PBS-treated group served as the control. (B) Bioluminescence images of mice treated with PD-1 scFv, TREM2 scFv, and BsAb at days 7, 14, and 21 (n = 4). (C) Bioluminescence signals of individual groups at day 7, 14, and 21 are shown on the Y-axis. The PBS group is indicated as black, the PD-1 scFv group is in orange, the TREM2 scFv group is in blue, and the BsAb group is in red. (D-E) Tumor volume and body weight of mice subjected to the indicated treatments (n = 4). (F-H) MC38-CEA tumors excised from individual groups of sacrificed mice on day 23. Tumor volume and weight of mice subjected to the indicated treatments (n = 4). (I) Effect of different treatments on the survival of tumor-bearing mice. (J-L) Effect of different treatment modalities on the infiltration of CD8-positive T cells (J), MDSCs (K), and M2 TAMs (L). Proportions of tumor-infiltrating CD8-positive T cells, MDSCs, and M2 TAMs were detected by flow cytometry on day 23. All cells gated on CD45, CD45 + CD11b Gr1 + were marked as MDSCs, whereas cells gated at CD45 + CD11b F4/80 CD206 were marked as M2 TAMs. Representative plots are shown on the right (n = 4). Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by unpaired Student’s t test and one-way ANOVA. scFv, single-chain fragment variable; CEA, carcinoembryonic antigen; PD-1, programmed death-1; TREM2, triggering-receptor-expressed on myeloid cells 2; PBS, phosphate buffered saline; MDSCs, myeloid-derived suppressor cells; TAMs, tumor associated macrophages; CD, cluster of differentiation