Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: A. Schematic outline of the prevention experiment. TCL1-192 cells were injected on d0 and 200μg mouse αSlamf6 (13G3) or a mouse IgG2a isotype control was injected i.p. into SCID mice on day 7, 14 and 21. Mice were sacrificed on day 28. B. Spleen size and weight at day 28. Administering αSlamf6 vs IgG2a isotype caused a 5.0- fold reduction (0.15 ± 0.02 vs. 0.78 ± 0.08 g; p = 0.0003 ) or 5.8-fold reductions comparing αSlamf6 vs. no antibody (0.15 ± 0.02 vs. 0.87 ± 0.02 g; p < 0.0001 ). C. A 26-fold reduction of the number of TCL1-192 cells was detected in the spleen of αSlamf6-injected vs. non-injected (1.7 ± 0.8 × 10 7 vs. 4.4 ± 0.3 × 10 8 p < 0.0001 ); or 29-fold in αSlamf6-injected vs. isotype-injected (1.7 ± 0.8 × 10 7 vs. 4.9 ± 0.7 × 10 8 ; p = 0.0008 ) SCID mice. Cells were gated on viable, DAPI − , B220 + CD5 + cells. Counting beads were used to determine the number of TCL1-192 cells per μl of blood. D. A 113-fold reduction of TCL1-192 cells in the blood of αSlamf6-injected vs. non-injected mice was found (0.3 ± 0.1 × 10 3 vs. 3.4 ± 0.4 × 10 4 per μl blood; p = 0.0003 ); 100-fold in αSlamf6-injected vs. isotype-injected mice (0.3 ± 0.1 × 10 3 vs. 3 ± 1.1 × 10 4 per μl blood; p = 0.047) . E. Number of TCL1-192 cells in the peritoneal cavity: αSlamf6-injected vs. non-injected (9.38 ± 3.6 × 10 6 vs. 5.8 ± 2.3 × 10 6 ) or αSlamf6-injected vs. isotype-injected (9.38 ± 3.6 × 10 6 vs. 1 ± 0.1 × 10 7 ). F. Number of TCL1-192 cells in the omentum: αSlamf6-injected vs. non-injected (9.5 ± 1.55 × 10 6 vs. 5.9 ± 1.2 × 10 6 or αSlamf6-injected vs. isotype-injected (9.5 ± 1.55 × 10 6 vs. 8.3 ± 0.7 × 10 6 ). Results are representative of at least 3 independent experiments.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Injection, Control

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: A. Expression of Slamf6 on CD19 + CD20 + LMP2A/lMyc cells. B. Schematic outline of the experiment. Rag1 −/− mice were i.p. injected with 1 × 10 6 cells and injected i.p. with 200 μg αSlamf6 or isotype control on day 7 and 14. Mice were sacrificed on day 19. C. Differences in spleen size and weight on day 19. A 3-fold reduction was observed in spleen weight of mice injected with αSlamf6 compared to isotype-injected group (0.066 ± 0.005 vs. 0.15 ± 0.01; p = 0.0008). D. Percentage of CD19 + CD20 + LMP2A/λMyc tumors and absolute cell numbers in the spleen are shown. A 4-fold reduction in tumor burden was observed in αSlamf6-injected group (1.05 ± 0.17 × 10 6 vs. 4.07 ± 0.22 × 10 6 ; p < 0.0001). Representative of 2 independent experiments.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Expressing, Injection, Control

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: A. Three weeks after transplanting TCL1-192 cells, SCID mice were injected with αSlamf6 or isotype mIgG2a once a week (Squares indicate the number of injections). Mice were monitored everyday. Whereas isotype treated mice died between 35-42 days, αSlamf6 injected mice were still alive on day 60, when the experiment ended (n = 5 mice each group). B. Schematic outline of the experiment indicating the timeline of i.p. injection of TCL1-192 cells and i.p . injections of 200 μg αSlamf6 (13G3) or the IgG2a isotype control on day 21 and 28. Mice were monitored by bleeding on days 21, 24 and 27 and were sacrificed on day 35. C. A 2.5-fold reduction in spleen size was caused in αSlamf6 vs. isotype treated mice (0.55 ± 0.03 vs. 1.4 ± 0.08g; p = 0.0018) or 2.4-fold vs. untreated mice (1.303 ± 0.13 g; p < 0.0001 ) on day 35. The number of TCL1-192 cells was 3-fold reduced in αSlamf6 vs. isotype treated mice (3.4 ± 0.6 × 10 8 vs. 1.1 ± 0.1 × 10 9 ; p = 0.005 ) or 4-fold vs. untreated mice (3.4 ± 0.6 × 10 8 vs. 1.36 ± 0.1 × 10 9 p = 0.0002 ). D. The number of TCL1-192 cells in the peritoneum of αSlamf6 injected and isotype or non-injected SCID mice (3 ± 0.5 × 10 8 vs. 2.3 ± 0.5 × 10 8 and 3.1 ± 0.6 × 10 8 p = 0.8 and p = 0.4 ). The data are representative of 3 independent experiments. Results are representative of 4 independent experiments.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Injection, Control

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: Schematic outline of the experiment is described in Figure 3B. A. Left Panel: Representative flow cytometry plot showing the overlay of B220 + CD5 + cells from the peritoneum, blood and spleen. While TCL1-192 cells from the peritoneum, spleen and blood express comparable levels of high B220 and CD5; the blood contains a second population that expresses lower levels of B220 and CD5 (named B220 int CD5 int ). Right Panel: Percentage of B220 + CD5 + TCL1-192 cells in the blood of SCID mice at weeks 3, 4 and 5 in non-injected and αSlamf6 or mIgG2a injected groups. Representative gating for the two sub-populations (B220 int CD5 int and B220 hi CD5 hi ) is depicted in the lower right corner for the isotype control group. B. On day 28, the total B220 + CD5 + cell number per μl blood is a 100-fold less in αSlamf6-injected vs. mIgG2a injected mice (per μl blood: 0.6 ± 0.1 × 10 3 vs. 6 ± 1 × 10 4 ; p = 0.001 ); or 85-fold less as compared to the non-injected group (0.6 ± 0.1 × 10 3 vs 5.1 ± 0.6 × 10 4 ; p = 0.0002 ). Although cells keep expanding by day 35, αSlamf6 injected group still has significantly less leukemic burden compared to mIgG2a injected (per μl blood: 7.5 ± 2.1 × 10 4 vs. 4.9 ± 0.7 × 10 5 ; p = 0.0015 ) vs. non-injected group (6.1 ± 0.7 × 10 5 ; p = 0.0006 ). The data are representative of 3 independent experiments. C. Number of B220 hi CD5 hi leukemic cells in the blood of αSlamf6 treated mice is less than that in non-treated mice (0.63 ± 0.19 × 10 5 vs. 1.2 ± 0.1 × 10 5 ; p = 0.02). D. Mean Fluorescence Intensity (MFI) values of B220 and CD5 ex pression on B220 hi CD5 hi and B220 int CD5 int populations of the non-treated group at week 5. Results are representative of 4 independent experiments.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Flow Cytometry, Injection, Control, Fluorescence

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: Schematic outline of the experiment is described in . A. Comparison of total B220 + CD5 + cells in blood of the non-treated group (−) to its B220 hi CD5 hi subpopulation and B220 hi CD5 hi TCL1-192 cells in the αSlamf6-treated (+) mice by Annexin-V/7-AAD staining. B. MFI values of surface IgM (sIgM) and CXCR4 expression on B220 hi CD5 hi and B220 int CD5 int TCL1-192 cells in the blood of αSlamf6-treated (+) and non-treated (−) mice. C. Proximal BCR signaling in B220 hi CD5 hi and B220 int CD5 int populations in the blood of αSlamf6-treated (+) and non-treated (−) mice was assessed by intracellular staining of pSyk, pBtk and pPLCγ2 and measured by flow cytometry. Representative of three experiments. D-F. Expression of pSyk, pBtk and pPLCγ2 in spleen and peritoneum. Results are representative of 4 independent experiments.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Comparison, Staining, Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: F(ab') 2 αSlamf6 was generated using Pierce F(ab') 2 preparation kit. A. Outline of the experiment. TCL1-192 cells were i.p. injected on d0. On d7, d14 and d21, 200 μg αSlamf6 (13G3) was i.p. injected, or on d7, d9, d14, d16, d21 and d23, 200 μg F(ab') 2 αSlamf6 was injected. Mice were sacrificed on d28. B. Differences in spleen size and weight in non-injected, αSlamf6 or F(ab') 2 αSlamf6 injected SCID mice on d28. C-E. Representative staining of B220+CD5+ from non-injected mice and number of B220+CD5+ cells in SCID mice after αSlamf6, F(ab') 2 αSlamf6 injected were compared to those of non-injected mice in spleen, blood and peritoneum. Next, levels of pSyk F. , pBtk G. and pPLCγ2 H. in TCL1-192 cells isolated on d28 from non-injected and F(ab') 2 αSlamf6 injected mice were compared. Representative of 2 independent experiments with n = 9 mice total for F(ab') 2 αSlamf6 injected group. P values are as shown.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Generated, Injection, Staining, Isolation

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: A. Outline of the experiment. TCL1-192 cells were i.p . injected on d0. On d21, group of mice were either i.p . injected 200 μg αSlamf6 (13G3), given 0.16 mg/ml ibrutinib in drinking water or was given both. Mice on ibrutinib received the drug until sacrifice. Mice received another injection of αSlamf6 on d28. Mice were sacrificed on d28. B. Differences in spleen size is as shown. C. Mice treated with a combination of αSlamf6 and ibrutinib had significantly smaller spleens and leukemic burden compared to αSlamf6-treated mice (weight: 0.31 ± 0.02 vs. 0.51 ± 0.04 g; p = 0.005 and a two-fold smaller number of TCL1-192 cells: 0.78 ×10 8 ± 1.54 × 10 7 vs. 1.53 × 10 8 ± 1.76 × 10 7 ; p = 0.01 ) on day 35. D. Relative numbers of TCL1-192 cells in the peritoneal cavity. BTK inhibitor resulted in 5.4 fold reduction in leukemic burden in peritoneal cavity compared to the control or αSlamf6 treated group alone (7.07 ± 1.43 × 10 7 vs. 3.82 ± 0.24 × 10 8 ; p < 0.0001). E. Representative FACS plots of B220 + CD5 + in different groups and the exact cell numbers are as shown in the graph below. Ibrutinib alone treated mice resulted in 3-fold lower leukemic burden in blood compared to non-treated mice (per μl blood: 2.2 × 10 5 ± 2.9 × 10 4 vs. 7.3 × 10 5 ± 7.7 × 10 4 ; p = 0.0003 ). However, αSlamf6 alone treated mice had significantly less TCL1-192 burden compared to ibrutinib alone group (0.67 × 10 5 ± 1.8 × 10 4 ; p = 0.0001 ). Difference in leukemic burden between αSlamf6 alone and ibrutinib/αSlamf6 treated group did not reach statistical significance (0.67 × 10 5 ± 1.8 × 10 4 vs. 0.27 × 10 5 ± 0.5 × 10 4 ; p = 0.06 ). F. Percentage of TCL1-192 cells in the bone marrow. Anti-Slamf6 resulted in 4.6- fold reduction in percentage of TCL1-192 cells in bone marrow compared to non-treated group (52.2 ± 4.4 vs. 11.2 ± 1.2%; p < 0.0001 ). When compared, αSlamf6 in combination with ibrutinib resulted in a further reduction (2 fold) compared to αSlamf6 treatment alone (11.2 ± 1.2 vs. 5.6 ± 0.8%; p = 0.006 ). Representative of 2 independent experiments is shown. P values are as shown.

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Injection, Control

Journal: Oncotarget

Article Title: A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells

doi: 10.18632/oncotarget.8378

Figure Lengend Snippet: A. 10 7 MEC-1 cells were subcutaneously injected into Rag2 −/− γc −/− mice. 200μg/ml mouse αhuman-SLAMF6 or isotype mIgG2b was injected i.p. on day 7 and 14 ( Left Panel ). Tumor volume was determined by measuring 3 diameters at indicated time points ( Right Panel ). B-C. OSU-CLL cells were cultured in vitro in the presence of 50 μg/ml Fc-crosslinker alone, 10 μg/ml mouse αhSLAMF6 and Fc-crosslinker, 0.5 μM ibrutinib or in combination for 72 hours and apoptosis was measured by Annexin-V+/PI-. B. The plots demonstrate the representative gating strategy for AnnexinV/PI staining. Results are representative of 3 independent experiments. (*: p < 0.05, **: p < 0.01, ***: p < 0.005)

Article Snippet: Mouse anti-human SLAMF6 antibody (994.1) was obtained from ARCA Biopharma (Westminster, CO).

Techniques: Injection, Cell Culture, In Vitro, Staining