Fig 2: AN3025 significantly inhibits MC38 tumor growth as a monotherapy in hTNFR2 mouse model. (A–E) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm3. (A, B) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg or 1mg/kg every 4 days intraperitoneally for 5 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) MC38 tumor bearing TNFR2 humanized mice were treated with AN3025 at the dosage of 10mg/kg, 3mg/kg every 3 days intraperitoneally for 3 doses in total. Tregs (CD45+CD3+CD4+ Foxp3+) frequency in total CD4+ T cells in the MC38 tumors was quantified by flow cytometry (n=6 each group). (D, E) MC38 tumor bearing TNFR2 humanized mice were treated intraperitoneally with 10mg/kg AN3025 alone or combination of 10mg/kg AN3025, 0.3mg/mouse anti-mouse CD4 depletion antibody and 0.3mg/mouse anti-mouse CD8 depletion antibody every 4 days for 5 doses in total. (D) Tumor volume measurement during the treatment (n=8 each group). (E) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.

Fig 3: AN3025 inhibits TNFa-TNFR2 mediated cell death of hTNFR2 overexpressing Jurkat cell and enhances T effector cell function in Tregs/Teff co-culture assay. (A) Expression of human TNFR2 on wildtype Jurkat cells. (B) Viability of wildtype Jurkat cells after stimulation with titrated human TNFa. (C) Expression of human TNFR2 on established hTNFR2 overexpressing Jurkat single cell clone. (D) Viability of hTNFR2 overexpressing Jurkat cells after stimulation with titrated human TNFa. (E) Viability of hTNFR2 overexpressing Jurkat cells after stimulation with constant 0.5ng/mL human TNFa in the presence of titrated AN3025 or titrated human IgG1, ?. (F) Competitive ELISA binding assay of AN3025 with biotinylated human TNFa to human TNFR2 on plate. (G, H) Human iTregs were co-cultured with CFSE-labeled autologous CD4+ Teff cells for 4 days. (G) CFSElo T effector cell percentage was quantified by flow cytometry. (H) IFN? in the supernatants was detected by ELISA assay. Values were expressed as Mean ± SEM. **P < 0.01; ***P < 0.001.

Fig 4: AN3025 enhances anti-tumor efficacy of mouse PD-1 antibody in combination study. (A–H) Murine colon cancer MC38 cells (5E5) were implanted subcutaneously into homozygous TNFR2 humanized mice (female). Mice were grouped when tumor volume reached approximately 100 mm3. (A–F) MC38 tumor bearing TNFR2 humanized mice were treated with 10 mg/kg AN3025 or 10mg/kg mouse PD-1 antibody (mPD-1 Ab) every 3 days intraperitoneally for 7 doses in total. (A) Tumor volume measurement during the treatment (n=8 each group). (B) Body weight record during the treatment (n=8 each group). (C) Representative immunohistochemistry images of CD8 staining on MC38 tumors. (D) Quantifications of CD8+ T cells per mm2 by immunohistochemistry (n=3 each group). (E) Representative immunohistochemistry images of CD4 staining on MC38 tumors. (F) Quantifications of CD4+ T cells per mm2 by immunohistochemistry (n=3 each group). (G, H) MC38 tumor bearing TNFR2 humanized mice were treated with 3mg/kg AN3025 or 3mg/kg mPD-1 Ab or combination of AN3025 and mPD-1 Ab every 3 days intraperitoneally for 7 doses in total. (G) Tumor volume measurement during the treatment (n=8 each group). (H) Body weight record during the treatment (n=8 each group). Values were expressed as Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.