Anti-Human CTLA4 Recombinant Antibody (Ticilimumab (Tremelimumab)) (CAT#: TAB-206)

Figure 1 Binding profiles of ipilimumab-scFv/CTLA-4 and tremelimumab-scFv/CTLA-4 measured by SPR.

CTLA-4 was immobilized on the chip while serially diluted (A) ipilimumab-scFv or (B) tremelimumab-scFv with concentrations ranging from 6.25 nM to 200 nM were then flowed through the chip and the response units were measured.

He, M., Chai, Y., Qi, J., Zhang, C. W., Tong, Z., Shi, Y., ... & Gao, G. F. (2017). Remarkably similar CTLA-4 binding properties of therapeutic ipilimumab and tremelimumab antibodies. Oncotarget, 8(40), 67129.

Figure 2 Modulation of CTLA4 and phosphoprotein expression in monocytes exposed ex vivo to tremelimumab.

Monocytes were cultured in increasing concentrations of tremelimumab for 48 hours, after which were analyzed for intracellular flow cytometry. Data is presented as fold change from the baseline mean fluorescence intensity (MFI) at increasing concentrations of tremelimumab (0, 0.1, 1. 10. 15 and 100 mg/ml) presented in a semilogarithmic plot. Red triangles represent monocytes samples obtained from three patients with metastatic melanoma (MD: melanoma donors); black circles are results from monocytes analyzed from five healthy subjects (HD: healthy donors). a) Intracellular CTLA4 expression; b) pp38(pT180/pY182); c) pErk1/2 (T202/204); d) pAkt (pT308); e) pSTAT1(pY701); f) pSTAT3 (pY705); g) pSTAT5 (Y694); h) pSTAT6 (Y641). **p<0.01; ***p<0.001.

Comin-Anduix, B., Sazegar, H., Chodon, T., Matsunaga, D., Jalil, J., von Euw, E., ... & Koya, R. C. (2010). Modulation of cell signaling networks after CTLA4 blockade in patients with metastatic melanoma. PloS one, 5(9), e12711.

Figure 3 Immune response against HCV antigens after tremelimumab.

(A) IFN-c producing T lymphocytes against HCV antigens were evaluated by ELISPOT assays at different time points after treatment. Antigens used were recombinant core, NS3, NS4, and NS5 proteins, and peptide pools spanning the whole HCV polyprotein. Results represent mean + SEM of spot forming cells (SFC). For peptides, values correspond to the sum of the different pools encompassed in each protein. (p <0.05 compared to day 0). (B) Representative example of anti-HCV responses obtained in patients B and F.

Sangro, B., Gomez-Martin, C., de la Mata, M., Iñarrairaegui, M., Garralda, E., Barrera, P., ... & Lasarte, J. J. (2013). A clinical trial of CTLA-4 blockade with tremelimumab in patients with hepatocellular carcinoma and chronic hepatitis C. Journal of Hepatology, 59(1), 81-88.

Figure 4 Effect of tremelimumab on the suppressive activity of Tregs in vitro.

CD4+ T cells were positively selected from PBMC samples using CD4 microbeads. CD4+ T cells were labeled with antibodies and used to sort CD4+ CD25+ CD127− Tregs and CD4+ CD25− CD127+ Teff by FACSAria. Tregs and Teff were cultured in triplicate wells alone or together at the ratios indicated with HLA-DR+ cells as APCs and anti-CD3/28 antibodies in the absence or presence of 15 μg/ml tremelimumab. Proliferation was measured by tritiated thymidine and reported as CPM (A). The percent suppression of Teff proliferation by Tregs was also determined (B). Data shown are derived from one donor and representative experiments from 3 different donors.

Khan, S., Burt, D. J., Ralph, C., Thistlethwaite, F. C., Hawkins, R. E., & Elkord, E. (2011). Tremelimumab (anti-CTLA4) mediates immune responses mainly by direct activation of T effector cells rather than by affecting T regulatory cells. Clinical Immunology, 138(1), 85-96.

Figure 5 Representative immunohistochemistry of inspiratory muscles at autopsy, 66 days after tremelimumab-durvalumab treatment.

Hematoxylin and eosin (H&E) sections show inflammatory myopathy in the diaphragm and intercostal muscles without rimmed vacuoles and without perifascicular atrophy, consistent with polymyositis. A mononuclear infiltrate is present which invades otherwise normal myofibers and completely effaces the background muscle fiber architecture in some areas. ATPase shows intense staining in small fibers compared to surrounding lighter, normal sized fibers in preserved areas of muscle, indicative of type II fiber atrophy. Trichrome shows mildly increased connective tissue, but shows no rimmed vacuoles, rods, or other inclusions. T cell co-receptor staining (CD3, CD4, CD8) revealed a mixed T-cell infiltrate which often completely effaced the myofascicular architecture. CD68 highlights necrotic myofibers scattered within the larger inflammatory infiltrate. PD-L1 expression was observed in blood vessels of dying muscle. Weak CTLA-4 expression was detected in necrotic myofibers. All images are 100× magnification.

John, S., Antonia, S. J., Rose, T. A., Seifert, R. P., Centeno, B. A., Wagner, A. S., & Creelan, B. C. (2017). Progressive hypoventilation due to mixed CD8+ and CD4+ lymphocytic polymyositis following tremelimumab-durvalumab treatment. Journal for immunotherapy of cancer, 5(1), 54.

Figure 1 Anti-Human CTLA4 Antibody (TAB-206) in SDS-PAGE

SDS-PAGE analysis of TAB-206 in non-reduced (lane 1, 2.5ug) and reduced (lane 2, 2.5ug) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

Figure 2 Anti-Human CTLA4 Antibody (TAB-206) in HPLC

The purity of TAB-206 was greater than 95% as determined by SEC-HPLC.