Afuco™ Anti-Human TNFRSF13C ADCC Recombinant Antibody (VAY736), ADCC Enhanced (CAT#: AFC-605CL)

Figure 1 VAY-736 engages surface molecules on CLL B cells and NK cells.

Flow cytometry analysis of VAY-736 binding profile on different cell subsets enriched from primary CLL or healthy donor blood. Peripheral blood mononuclear cells are stained with fluorochrome- conjugated VAY-736, anti-BAFF-R (11C1), isotype control, or VAY-736 variant with null Fc binding (N297). (A) Four CLL B-cell samples, IGHV mutated (M) or unmutated (UM) (CD45+, CD19+, and CD5+) stained with VAY-736 (blue) and anti-BAFF-R (11C1) (red) compared with isotype control (purple). (B) Patients ΔMFI stratified according to IGHV mutational status (P <.05 mutated vs unmutated; mutated, n = 6; unmutated, n = 10; 2-sample Student t test). (C) Representative flow cytometry analysis of VAY-736 or anti-BAFF-R binding to healthy T cells (CD45+, CD3+, and CD19-; n = 10). (D) Representative flow cytometry analysis of VAY-736 and N297 binding to CLL NK-cells (CD45+, CD56+, and CD3-; n = 8). *P <.05.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.

Figure 2 BAFF-mediated survival is blocked by VAY-736 treatment of CLL B cells.

(A) Representative annexin V/propidium iodide (PI) flow cytometry analysis of a CLL patient B-cell viability at 72 hours. (B) Time course comparison of primary CLL cells' viability at 24, 48, and 72 hours treated with BAFF (500 ng/mL), VAY-736, or cells pretreated with VAY-736 subsequently stimulated with BAFF (P <.001: 72 hours, BAFF vs untreated; P <.01: 48 hours, VAY-736 + BAFF vs BAFF). Inhibitory effect of VAY-736 at each time point was tested by interaction contrast (BAFF + VAY-736 subtracting BAFF vs VAY-736 subtracting untreated; 72 hours, P <.01). Data were analyzed by using a mixed effect model, and Holm's method was used to adjust multiplicity (n = 21 patients). (C-E) Western blot analysis of p100 and p52 protein levels in primary CLL patient B cells. Treatments were soluble BAFF (500 ng/mL) for 16 hours with or without pretreatment with VAY-736 (10 μg/mL). Activation of alternative NF-κB was determined by separate cytoplasmic (CE) and nuclear (NE) protein fractions, loading controls actin (cytoplasmic), and Lamin B (nuclear). Quantification of p100 or p52 protein by densitometry analysis was normalized to loading control and then to the untreated condition (P <.01: cytoplasmic p100, VAY-736 + BAFF vs BAFF; P <.05: p52 cytoplasmic and nuclear levels, BAFF vs untreated, VAY-736 + BAFF vs BAFF; n = 5 CLL patients, 3 independent experiments). *P <.05, **P <.01, and ***P <.001.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.

Figure 3 VAY-736 blocks BAFF-mediated activation of NF-κB at the single-cell level.

(A) OSU-CLL cells were transduced with Cignal-GFP-NF-κB Lenti-viral reporter construct (Qiagen, Hilden, Germany). Cells were treated with VAY-736 with or without 16-hour BAFF stimulation (500 ng/mL). GFP-NF-κB activity (488 nm) was measured via fluorescent microscopy. Analysis of GFP-NF-κB activity was performed by using NIS-Elements software (Nikon Instruments, Tokyo, Japan). A representative image from 5 independent experiments is shown. (B) Data are expressed as relative florescence unit (RFU) mean fold change ± standard error of the mean (SEM) relative to untreated control. Mean background fluorescence was subtracted from single OSU-CLL cells that were transfected with the negative control construct. The data represent 5 independent experiments. A one-way analysis of variance followed by Bonferroni post hoc analysis was performed to determine statistical significance between groups (n = 435 single cells). (C) Representative 14-hour time lapse of single OSU-CLL cell GFP-NF-κB activity. GFP-NF-κB levels in VAY-736-treated cells were comparable to untreated control cells (data not shown). (D) Compiled 14-hour time lapse data of single cells monitored for GFP-NF-κB activity (488 nm) expressed as relative florescence unit mean fold change ± SEM relative to untreated control (n = 48 single cells,>3 independent experiments) treated with BAFF (500 ng/mL), pretreatment of VAY-736 (10 μg/mL), or untreated. **P <.01 and ***P <.001.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.

Figure 4 BAFF stimulates NF-κB signaling in CLL treated with ibrutinib.

(A-B) Western blot and densitometry analysis of p100 and p52 protein levels in primary CLL patient B-cell cytoplasmic-enriched protein lysates. Treatments with ibrutinib (1 μM) or dimethyl sulfoxide (vehicle) control for 1 hour and washed out, and followed by 16 hours of vehicle, BAFF (500 ng/mL), VAY-736 (10 μg/mL), or VAY-736 + BAFF. (C-D) OSU-CLL cells transduced with Lenti-viral Cignal-GFP-NF-κB reporter were pretreated with ibrutinib (1 μM) or dimethyl sulfoxide vehicle control followed by treatments of BAFF (500 ng/mL), VAY-736 (10 μg/mL), or VAY-736 + BAFF. GFP-NF-κB activity was measured via fluorescent microscopy 14 hours posttreatment. A representative image is shown (n = 106 single cells, 3 independent experiments). Data are expressed as the mean RFU fold change ± SEM relative to the vehicle control. A one-way analysis of variance followed by Bonferroni post hoc analysis was performed to determine whether statistical significance existed between groups. *P <.05, **P <.01, and ***P <.001.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.

Figure 5 VAY-736 enhances ADCC and NK-cell activation.

(A) ̶⁵¹Cr release assays comparing ADCC mediated by VAY-736 with allogeneic normal donor NK cells and CLL target B cells (P <.001, n = 8 normal donor; n = 7 CLL). (B) Enzyme-linked immunosorbent assay data of IFN-γ release by NK cells incubated with plate-bound VAY-736 vs OBN at 10, 1.0, and 0.1 μg/mL; P <.05, n = 3 NKs, 2 separate experiments. (C-D) Enzyme-linked immunosorbent assay data comparing TNF-α release by monocytes (C) or MDMs (D) stimulated with plate-bound VAY-736, OBN, OFA, RTX, alemtuzumab (ALE), or immunoglobulin G (IgG) (not significant, n = 3). (E) MDMs were labeled with Claret, and CLL B cells were stained with PKH67. ADCP was observed by using flow cytometry and measuring the percentage of cells double-positive for Claret PKH67 (P <.05: untreated vs RTX, vs OBN, vs OFA, vs VAY-736; n = 3). *P <.05 and ***P <.001. TRA, trastuzumab.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.

Figure 6 In vivo efficacy of VAY-736 in the Eμ-Tcl1 mouse model of CLL.

(A) Histogram flow cytometry analysis of VAY-736 binding over isotype to CD5+ CD19+ double-positive Eμ-TCL1 mouse splenocytes. (B) Representative FACS of CD5+ CD19+ percent double-positive leukemia lymphocyte population in peripheral blood samples from a leukemia-burdened Eμ-TCL1 transgenic mouse. Mice were injected weekly for 2 weeks total with 100 mg/kg of VAY-736. Blood was collected 1 day after treatment and weekly. (C-D) Peripheral blood from Eμ-TCL1 mice were collected 24 hours before (Pre tx) and 24 hours after (Post tx) injection of VAY-736 at 100 mg/kg or phosphate-buffered saline (PBS) vehicle control, and CD5+ CD19+ double-positive leukemic cells were counted (C) and percentages were analyzed (D) by using flow cytometry. (E) Survival analysis of SCID mice engrafted with leukemia-burdened Eμ-TCL1 splenocytes from a single donor. SCID mice were enrolled in the study when CD5+ CD19+ percent lymphocytes from peripheral blood reached>20% within 9 weeks of engraftment. Mice received weekly injections of VAY-736 (10 mg/kg) or PBS vehicle control for 6 weeks (P =.0169: VAY-736; n = 3, vs vehicle, n = 4). (F) Eμ-TCL1 splenocytes from a highly leukemic burdened mouse were adoptively transferred into NOTAM mice. Peripheral leukemic percentages were monitored by using flow cytometry before engraftment, pretreatment, 24 hours posttreatment, and at 12 days posttreatment with either VAY-736 injection (10 mg/kg) or vehicle (PBS). (G) Mice from panel F were euthanized at day 12 posttreatment, and the leukemic burden of the spleen and bone marrow were analyzed according to percent CD5+ CD19+ by using flow cytometry.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.

Figure 7 VAY-736 combines effectively with ibrutinib in vivo.

Tumor-derived splenocytes from disease-burdened Eμ-TCL1 mice were engrafted into SCID mice. (A) Kaplan-Meier survival plot receiving weekly VAY-736 (10 mg/kg) retro-orbital injections, vehicle control injections, ibrutinib drinking water, or combination VAY-736 + ibrutinib, for up to 6 injections of VAY-736 or vehicle, and continuous ibrutinib drinking water provided through study. Leukemic death was confirmed upon death by fluorescence-activated cell sorting analysis of blood, spleen, and bone marrow CD5+ CD19+ percentage of lymphocytes (VAY-736 + ibrutinib vs ibrutinib, P <.0001; VAY-736 + ibrutinib vs VAY-736, P =.06; n = 14 per group). Adjustments for multiple comparisons for the log-rank test were used for data analysis. *P <.05 and **P <.01. (B) After 6 weeks, 2 animals per group were euthanized and samples sent for histopathology analysis. Splenic white pulp expanded by leukemia cells is visible as white foci in the enlarged spleens. Splenic white pulp areas (arrows) and red pulp (arrow heads) are infiltrated and expanded by leukemia cells in all specimens. Top row: photomicrographs at low-power magnification to show level of infiltration (magnification ×20; hematoxylin and eosin stain). Middle row: high-power magnification to show cellular detail (magnification ×60; hematoxylin and eosin stain). Mitotic figures are indicated with an asterisk. Bottom row: gross anatomy images of spleens. White blood cell (WBC) counts from blood smears (C) and CD5+ CD19+ percent circulating lymphocytes (D) were observed weekly for leukemia progression. (E-F) At death, spleen and bone marrow were analyzed by fluorescence-activated cell sorting for percent lymphocytes CD5+ CD19+. (G) Kaplan-Meier survival plot of a repeated experiment described in panel A except with engraftment into NOTAM mice lacking functional FcRγ (VAY-736 + ibrutinib vs ibrutinib, P =.4921; VAY-736 vs vehicle, P =.7453; n = 7 to 8 per group). NS, not significant.

McWilliams, E. M., Lucas, C. R., Chen, T., Harrington, B. K., Wasmuth, R., Campbell, A.,... & Awan, F. T. (2019). Anti–BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib. Blood advances, 3(3), 447-460.