Fig 2: ROR1/CD3 TCE shows high binding affinity to ROR1 and CD3. (A) The schematic diagram of ROR1/CD3 antibody. The green part is the Fab targeting ROR1, and the right yellow part is the single-chain variable fragment (scFv) targeting CD3. ROR1/CD3 TCE molecule consists of three chains: anti-ROR1 antibody heavy chain (chain 1), anti-ROR1 antibody light chain (chain2) and an anti-CD3 antibody ScFv-Fc fused chain (Chain 3). (B, C) Cell-based binding of ROR1/CD3 TCE molecule to ROR1-expressing MDA-MB-231 cell and CD3-expressing human T cells. Cells were incubated with a serially diluted ROR1/CD3 antibody followed by staining with PE-conjugated anti-human IgG. The MFI was determined by flow cytometry. The experiment was performed with three technological replicates and validated by other two biological replicates. (D) The binding affinity of ROR1/CD3 to ROR1 and CD3 was evaluated by the surface plasma resonance. The experimemnt was performed with three technological replicates. KD, binding dissociation equilibrium constants; Ka, kinetic association rate; Kd, kinetic dissociation rate

Fig 3: ROR1/CD3 TCE induced T cell activation in a ROR1-dependent manner. (A, B) The activation of CD4+, CD8+ T cells were determined by flow cytometry via analyzing the activation marker CD25 and CD69. (C, D) The proliferation of CD4+ and CD8+ T cells was measured after co-cultured with a serially diluted ROR1/CD3 antibody for 96 h. The experiment was performed with three tecnological replicates, and validated by another PBMC donor

Fig 4: ROR1/CD3 inhibited tumor growth in TNBC xenograft mouse model. (A) The schematic diagram for the in vivo efficacy study (left panel). Administration of 1 mpk ROR1/CD3 antibody induced significantly tumor growth of NOG mice bearing MDA-MB-231 compared with mice treated with control IgG (right panel). N = 8 mice for each group. Two-way ANOVA was performed for data analysis. (B) No body weight loss was observed within mice treated with ROR1/CD3 antibody. (C, D) ROR1/CD3 suppressed the tumor growth of HCC1937 model on NOG mice. N = 8 mice for each group. Two-way ANOVA was performed for data analysis (C). No significant body weight loss was observed with the administration of ROR1/CD3 molecule (D). ROR1/CD3 engaged tumor-infiltrating T cells. Mice were treated with 1 mpk ROR1/CD3 after 20 days’ of tumor implantation. At day 27, tumors were isolated and analyzed for T cell activation and infiltration by flow cytometry. The percentage of CD4+, CD8+ T cells in tumors (E), the ratio of CD4+ or CD8+ in CD45+ T cell populations (F), granzyme B+ positive CD4+/CD8+ in CD4+ or CD8+ T cell (G), the ratio of CD4+ or CD8+ T cells in CD45+ population of the spleen (H) was determined. S student’s t test was performed for data analysis

Fig 5: ROR1/CD3 TCE killed ROR1-expressing TNBC cells. (A) The cytotoxic lysis of ROR1/CD3 antibody to TNBC cells were detected by measuring the lactate dehydrogenase release. (B-G) Human PBMCs were incubated with ROR1/CD3 antibody molecule and the cytokine release in the supernatants was detected by ELISA. Both the cytotoxic and cytokine release experiments were performed with three tecnological replicates, and validated by another PBMC donor