Journal: Antibodies

Article Title: CEA-4-1BBL: CEACAM5-Targeted 4-1BB Ligand Fusion Proteins for Cis Co-Stimulation with CEA-TCB

doi: 10.3390/antib14040096

Figure Lengend Snippet: CEA-4-1BBL-mediated tumor growth inhibition in humanized mice ( A , B ) The human CEACAM5 expression and human CD3+ cell infiltration measured by IHC on FFPET at a tumor size of 200 mm 3 before treatment of MKN-45 or HPAFII tumors are shown. ( C ) CD34+ human cord blood humanized NOG mice received a subcutaneous injection of MKN-45 tumor cells. When a tumor volume of around 200 mm 3 was reached, the mice were randomized and treated with the vehicle (gray filled circle) or 2.5 mg/kg CEA-TCB (black filled diamond) twice per week. Four of the six groups received a combination treatment of 2.5 mg/kg CEA-TCB twice per week and 3 mg/kg CEA-4-1BBL once per week (the different colors indicate CEA-4-1BBL molecules implementing different CEACAM5 binders). The schedule of treatment is indicated along the x-axis; tumor growth in mm 3 is indicated along the y-axis. Each point indicates the mean for 10 mice; error bars show the standard error of the mean (SEM). ( D ) A repetition of the experiment shown in C with indicated molecules. ( E ) A repetition of the experiment shown in C but with HPAFII tumor cells instead of MKN45 with indicated molecules. Statistical analysis was performed using 1-way analysis of variance (ANOVA), and significant p values are indicated as follows: ns: not significant; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Immunohistology ex vivo: Immunohistochemical staining was performed in formalin-fixed paraffin-embedded tissue (FFPET) as described in [ , ] with human CD8 (Cell Marque Tissue Diagnostic, Sigma-Aldrich, Buchs, Switzerland, clone SP16) and human CD3 (Thermo Fisher Scientific, Reinach, Switzerland, MA5-13473, clone C8/144B).

Techniques: Inhibition, Expressing, Injection

Journal: Antibodies

Article Title: CEA-4-1BBL: CEACAM5-Targeted 4-1BB Ligand Fusion Proteins for Cis Co-Stimulation with CEA-TCB

doi: 10.3390/antib14040096

Figure Lengend Snippet: CEA-4-1BBL mediated increased CD8 T cell accumulation in a dose-dependent manner. ( A ) CD34+ human cord blood humanized NOG mice received subcutaneous injection of MKN-45 tumor cells and murine 3T3 fibroblasts. When a tumor volume of around 200 mm 3 was reached, mice were randomized and were treated with vehicle (gray) or 2.5 mg/kg CEA-TCB (black) twice per week. Three of the five groups received a combination treatment of 2.5 mg/kg CEA-TCB twice per week and 1m 3 or 10 mg/kg CEA-4-1BBL once per week (different shades of green). The schedule of treatment is indicated along the x-axis; tumor growth in mm 3 is indicated along the y-axis. Each point indicates the mean of 10 mice; error bars show the standard error of the mean (SEM). ( B ) The human CEACAM5 expression measured by IHC on FFPET at a tumor size of 200 mm 3 before treatment is shown. ( C ) At the endpoint (day 44), tumors were isolated and weighted. Three to four of ten tumors were digested and analyzed by flow cytometry. The counts of CD45+ CD3+ T cells, CD45+ CD3+ CD4+ T cells and CD45+ CD3+ CD8+ T cells per mg tumor are shown. Each symbol indicates an individual mouse; the bars indicate the mean, and the error bars the standard deviation (SD). ( D ) A total of 3-6 tumors per group were harvested at the endpoint (day 44); the tissue was formalin-fixed, paraffin-embedded and stained by immunohistochemistry (IHC) for human CD8 to monitor the tumor infiltration of CD8+ T cells. Tissue sections were scanned, and whole scans were analyzed by Definiens. One representative picture per group is shown. ( E ) The CD3+ and CD8+ T cell counts per square millimeter of immunohistochemistry are shown. Each symbol represents one individual mouse; the bars indicate the mean, and the error bars indicate the standard deviation (SD). Significance was calculated using unpaired one-way ANOVA with Tukey’s multiple comparison test. ns: not significant; * = p ≤ 0.05; ** p < 0.01. ( F ) Tumor was digested and centrifuged at endpoint day 44. Human cytokines were measured from supernatant using a 24plex R&D. For vehicle, CEA-TCB and CEA-TCB + 3 mg/kg T84.66-LCHA-4-1BBL, tumors from n = 4 mice were analyzed; for CEA-TCB + 1 mg/kg T84.66-LCHA-4-1BBL, tumors from n = 5 mice were analyzed; and for CEA-TCB + 10 mg/kg T84.66-LCHA-4-1BBL, tumors from n = 3 mice were analyzed. Measured cytokines are shown in different colors as the mean ± SD per treatment group. Statistical analysis was performed using 1-way analysis of variance (ANOVA), and significant p values are indicated as follows: ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Immunohistology ex vivo: Immunohistochemical staining was performed in formalin-fixed paraffin-embedded tissue (FFPET) as described in [ , ] with human CD8 (Cell Marque Tissue Diagnostic, Sigma-Aldrich, Buchs, Switzerland, clone SP16) and human CD3 (Thermo Fisher Scientific, Reinach, Switzerland, MA5-13473, clone C8/144B).

Techniques: Injection, Expressing, Isolation, Flow Cytometry, Standard Deviation, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Comparison