Afuco™ Anti-Human TNFSF9 ADCC Recombinant Antibody (PF-05082566), ADCC Enhanced (CAT#: AFC-618CL)

Figure 1 Octet competitive binding assay of the binding of 4-1BBL or utomilumab to mono- and di-4-1BB.

AR2G sensors loaded with mono- (left) or di- (right) 4-1BB were first saturated with 4-1BBL, utomilumab, or buffer. Then, the capacity of additional binding was monitored by measuring further shifts after the injection of additional 4-1BBL in the presence of saturated utomilumab, or vice versa.

Li, Y., Tan, S., Zhang, C., Chai, Y., He, M., Zhang, C. W. H.,... & Liu, W. J. (2018). Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab. Cell Reports, 25(4), 909-920.

Figure 2 Competition of utomilumab with 4-1BBL for the binding to mono- or di-4-1BB in flow cytometry assay.

Mono- or di-4-1BBs were incubated with different molar ratios of utomilumab before staining with 293T cells expressing 4-1BBLs.

Li, Y., Tan, S., Zhang, C., Chai, Y., He, M., Zhang, C. W. H.,... & Liu, W. J. (2018). Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab. Cell Reports, 25(4), 909-920.

Figure 3 SPR assay-based characterization of the binding of mono- (E) or di-4-1BB (F) to 4-1BBL, and mono- (G) or di-4-1BB (H) to utomilumabscFv, was accomplished using a single-cycle BIAcore T100 system.

The association constant (Ka), dissociation constant (Kd), and equilibrium dissociation constant (KD) of the binding are labeled accordingly. The fits of the binding curves are shown in red.

Li, Y., Tan, S., Zhang, C., Chai, Y., He, M., Zhang, C. W. H.,... & Liu, W. J. (2018). Limited Cross-Linking of 4-1BB by 4-1BB Ligand and the Agonist Monoclonal Antibody Utomilumab. Cell Reports, 25(4), 909-920.

Figure 4 Saturation binding of PF-05082566 to human and cynomolgus monkey 4-1BB.

Dose-dependent binding of AlexaFluor647-labeled PF-05082566 to a 300-19 cells transduced to express human 4-1BB, b 300-19 cells transduced to express cynomolgus monkey 4-1BB, c 72-h PHA-stimulated primary human PBMC, and d 72-h PHAstimulated primary cynomolgus monkey PBMC. The data are expressed as fold increase in geometric mean fluorescence intensity (Geo MFI) of cells stained with PF-05082566 relative to cells stained with the corresponding concentration of human IgG2 isotype control as measured by FACS. a, b display data from representative experiments with samples stained in duplicate. Error bars represent SEM. c, d represent data of binding to 72-h PHA-stimulated CD3- positive primary PBMC. Each line represents the staining result of cells isolated from individual donors.

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.

Figure 5 PF-05082566 induces NF-jB pathway activation through both human and cynomolgus monkey 4-1BB.

Dose-dependent activation of an NF-jB luciferase reporter in a 293T-cells-expressing human 4-1BB or b 293T-cells-expressing cynomolgus monkey 4-1BB. The data are representative of three separate experiments and shows fold increase in luciferase activity over unstimulated control cells by goat anti-huIgG Fc-specific F(ab')2 cross-linking antibody plus PF- 05082566 (black circles) or cross-linked human IgG2 isotype control (black triangles). Additionally, PF-05082566 treatment induces phospho-NF-jB p65 in anti-CD3-stimulated primary c human and d cynomolgus monkey CD3+ cells. The graphs demonstrate percentage of phospho-NF-jB p65+, 4-1BB+ T cells as measured by FACS. Cells were stimulated with CD3 alone (white bars), CD3 plus cross-linked 0.5 lg/ml PF-05082566 (gray bars), or CD3 plus cross-linked 5 lg/ml PF-05082566 (black bars). Significance relative to the control group was determined via two-tailed Student's t test (*p\0.05).

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.

Figure 6 PF-05082566 enhances IL-2 production by primary human T cells and expansion of antigen-specific CD8+ T cells in vitro.

a Dosedependent enhancement of IL-2 production as measured by ELISA from purified T cells stimulated with plate bound CD3 plus increasing concentrations of plate bound PF-05082566. Each line represents stimulation of purified T cells from one of seven individual donors tested in duplicate. b PBMC isolated from four separate HLA-A2+ donors whom had received an influenza vaccine within the prior season were stimulated in vitro with influenza matrix peptide (58-66, GILGFVFTL) for 8 days in the presence of either huIgG2 control antibody (white bars) or PF-05082566 (black bars). The data are represented as the percent viable CD8+, MHC HLA-A2-GILGFVFTL pentamer+ cells as measured by FACS. Significance was determined via 2-way ANOVA comparison with the control IgG2- treated group (**p\0.01, *p\0.05).

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.

Figure 7 PF-05082566 induces expansion of human CD45+ PBMC in vivo.

Engraftment of human PBMC adoptively transferred via intraperitoneal injection to NSG host mice was analyzed by FACS for human CD45 and Ki-67 staining 28 days following injection. On day 7 following PBMC injection, mice were treated with a single injection of human IgG2 isotype control or the indicated concentration of PF-05082566. a Mean percentage of human CD45+ lymphocytes assessed at several time points over the course of the study b percentage of human CD45+ lymphocytes at Study Day 28, 21 days following mAb injection and c percentage of human CD45+ Ki-67+ present in the whole blood samples of each treatment group at Study Day 28, 21 days following mAb injection. Data are representative of n = 7 mice per group and significance relative to the control group on the final day of the study was determined via two-tailed Student's t test (*p\0.05, **p\0.005)

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.

Figure 8 Intravenous administration of PF-05082566 to cynomolgus monkeys induces proliferation of CD8+ memory T cells.

a, b Multiple dose administration. Cynomolgus monkeys were injected intravenously with the indicated doses of PF-05082566 on days 1 and 8 of the study (n = 10 animals per group). On day 13, blood was collected, and cells were analyzed by flow cytometry. a The far left plot shows the gating strategy for identifying CD8+ TCM (CD95+ CD28+) and CD8+ TEM (CD95+ CD28-). The remainder of the plots shows representative staining for Ki-67 versus CD28 for CD8+ TCM (top row) and CD8+ TEM (bottom row) at the indicated dose levels. b Multiple dose administration of the indicated dose levels of PF-05082566 on day 1 and day 8 demonstrates significant increases in Ki-67+ CD8+ TCM cells (graph on left) and Ki-67+ CD8+ TEM (graph on right) as measured on study day 13.

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.

Figure 9 Intravenous administration of PF-05082566 to cynomolgus monkeys induces proliferation of CD8+ memory T cells.

c Single dose administration of 0.05 mg/kg (white bars), 1 mg/kg (gray bars), or 10 mg/kg (black bars) PF-05082566 to 2 individual animals per group demonstrates increases in Ki-67+ CD8+ TCM cells measured by FACS on study days 7 and 23 post dosing.

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.

Figure 10 PF-05082566 inhibits the growth of PC3 prostate carcinoma in vivo.

a Mean tumor volume at time points following subcutaneous administration of 3 million PC3 prostate carcinoma cells plus 1.5 million human PBMC in the right flank of SCID Bg mice (n = 8 animals/group). b The volume of each individual tumor on the final study day (day 21). Statistical significance was determined using 1-way ANOVA with Tukey's post-test (*p\0.05, **p\0.005).

Fisher, T. S., Kamperschroer, C., Oliphant, T., Love, V. A., Lira, P. D., Doyonnas, R.,... & Huang, C. (2012). Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunology, Immunotherapy, 61(10), 1721-1733.