Human Anti-KIR2DL3 Recombinant Antibody (clone 1-7F9) (CAT#: HPAB-0087-LSX)

Figure 1 Specificity of 1-7F9 antibody.

Characterization of 1-7F9 specificity for KIR2D subtypes. Transduced BWZ cells expressing individual KIR2DL or KIR2DS receptors were incubated for 30 minutes with anti-KIR antibodies (1 mg/ml), as indicated. The 1-7F9 was detected with PE anti–human IgG4, and EB6, GL183, and FES172 were revealed with PE goat anti–mouse IgG. Results shown are representative of 4 separate experiments.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 2 Specificity of 1-7F9 antibody.

Human whole blood from a healthy volunteer was stained with PEconjugated 1-7F9; dot plots represent 1-7F9 binding to the indicated leukocyte subsets based on forward/side light scatter.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 3 Specificity of 1-7F9 antibody.

Human whole blood from a healthy volunteer was stained with PEconjugated 1-7F9 and a combination of mAbs defining various leukocyte subsets, and analyzed by flow cytometry. Experiments in panels B and C have been performed on 11 healthy donors. Mean percentage and SD of 1-7F9–positive cells among the NK-and T-cell populations were 48.1% ± 14.9 and 2.4% ± 2.1, respectively.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 4 Specificity of 1-7F9 antibody.

Titration of 1-7F9 mAb on KIR2D-transduced BWZ cell lines. Cells were incubated for 30 minutes with 1/3 serial dilutions of 1-7F9, which were then revealed with PE anti–human IgG4 and analyzed by flow cytometry. Each dilution point was performed in duplicate. ◊, ■, ∆‚ and ● represent cell lines expressing KIR2DL1, KIR2DS1, KIR2DL3, and KIR2DS2, respectively. Mean and SD of data collected in 2 independent experiments are shown.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 5 Thawed human PBMC from KIR-S–positive (1 and 2) or KIR-S–negative (3 and 4) donors were incubated for 4 hours at 37°C, alone or in the presence of 1-7F9 (10 mg/ml), cognate isotypic control (IgG4, 10 mg/ml), or K562 (E:T ratio = 10) in the presence of anti-CD107 and monensin.

After incubation, cells were stained with anti-CD3 and anti-CD56, and then fixed, permeabilized, and stained with anti–IFN-γ. CD107 mobilization and IFN-γ production are then assessed on NK cells (CD3-CD56+ lymphocytes). Results are representative of 1 experiment of 2 done with a KIR-S–positive and a KIR-S–negative donor. Percentage of cells in each quadrant is shown.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 6 1-7F9 blocks interactions of inhibitory KIR2DL with HLA class I on B-EBV cells.

Binding of soluble KIR2DL1-hFc was blocked by anti-KIR mAbs GL183 or DF200 and binding of KIR2DL1-mFc blocked by 1-7F9, as measured by flow cytometry. Relative binding of KIR-Fc proteins to 221-Cw4 cells is shown as percentage of binding by KIR-Fc in the absence of mAbs. Similar data were obtained in a repeat experiment.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 7 1-7F9 blocks interactions of inhibitory KIR2DL with HLA class I on B-EBV cells.

In a ⁵¹Cr release cytotoxicity assay, YTS cells efficiently killed LCL721.221-Cw4 cells (▲), whereas YTS-2DL1 cells did not (●; E:T ratio 12:1). Preincubation (30 minutes at 37°C) of the NK cells with increasing doses of 1-7F9 augmented the killing of LCL721.221-Cw4 targets by YTS-2DL1 cells, in a dose-dependent manner. Curve fitting using one-site receptor saturation equation gives an EC50 of 0.71 mg/ml (95% CI, 0.2-1.2 mg/ml). Experiment shown is representative of multiple experiments giving equivalent results.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.

Figure 8 1-7F9–induced clearance of AML cells by human NK cells in NOD-SCID mice.

(A) Flow cytometric analysis of KIR and NKG2A expression by the polyclonal IL-2–activated NK-cell population that was used as effector cells in the in vitro cytotoxicity and the in vivo tumor rejection experiments shown in panels B and C, respectively. (B) □, Lysis of primary human AML cells by KIR ligand-matched NK cells, without (□) or with 1-7F9 antibody (■). (The NK cells were isolated from a healthy donor having the same HLA class I allotype groups as the AML target cells.) E:T ratio was 15:1. Results were analyzed by Student t test (***P <.005). (C) NOD-SCID mice infused with autologous NK cells and AML target cells at 1:3 E:T ratio died of leukemia within 65 days. Treatment with 1-7F9 (250 Mg/mouse) rescued mice challenged with NK and AML cells at an E:T ratio of 1:12, but not at an E:T of 1:18. N = 5 mice per group. Results have been analyzed by Kaplan Meier log rank test (***P <.005). Similar results were obtained in a repeat experiment.

Romagné, F., André, P., Spee, P., Zahn, S., Anfossi, N., Gauthier, L.,... & Della Chiesa, M. (2009). Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells. Blood, The Journal of the American Society of Hematology, 114(13), 2667-2677.