BAFF receptor antibody for mantle cell lymphoma therapy

Abstract

Mantle cell lymphoma (MCL) is an aggressive form of B cell non-Hodgkin's lymphoma and remains incurable under current treatment modalities. One of the main reasons for treatment failure is the development of drug resistance. Accumulating evidence suggests that B cell activating factor (BAFF) and BAFF receptor (BAFF-R) play an important role in the proliferation and survival of malignant B cells. High serum BAFF levels are often correlated with poor drug response and relapse in MCL patients. Our study shows that BAFF-R is expressed on both MCL patient cells and cell lines. BAFF-R knockdown leads to MCL cell death showing the importance of BAFF-R signaling in MCL survival. Moderate knockdown of BAFF-R in MCL cells did not affect its viability, but sensitized them to cytarabine treatment in vitro and in vivo, with prolonged mice survival. Anti-BAFF-R antibody treatment promoted drug-induced MCL cell death. Conversely, the addition of recombinant BAFF (rhBAFF) to MCL cells protected them from cytarabine-induced apoptosis. We tested the efficacy of a humanized defucosylated ADCC optimized anti-BAFF-R antibody in killing MCL. Our data show both in vitro and in vivo efficacy of this antibody for MCL therapy. To conclude, our data indicate that BAFF/BAFF-R signaling is crucial for survival and involved in drug resistance of MCL. Targeting BAFF-R using BAFF-R antibody might be a promising therapeutical strategy to treat MCL patients resistant to chemotherapy.

Keywords: Mantle Cell Lymphoma; adcc; baff; drug resistance; nk cells; therapy.

Figure 1.

BAFF-R antibody sensitizes MCL cells to chemotherapy. (a) Flow cytometry analysis on three liquid MCL patient samples (Pt1, Pt2, and Pt3). Black histograms, control isotype; red histograms, anti-BAFFR 11c1. (b) BAFF-R expression in three MCL cell lines JVM2, Jeko-1, and Mino cells by flow cytometry. Black histograms, control isotype; red histograms, anti-BAFFR 11c1. (c) 1 × 10⁵ Jeko-1 cells were seeded in 24-well plate in triplicates and cultured in the absence or presence of 200 ng/ml recombinant BAFF (rhBAFF) with or without 20 nM of cytarabine (CYT). The cell number was measured for the time indicated, not significant (ns) p > .05 for control Jeko-1 cells compared to rhBAFF-treated cells at day 5. ***p < .001 for Jeko-1 cells with cytarabine (CYT)+rhBAFF treatment compared with cells treated with CYT alone at day 7. One of the three experiments with triplicate samples shown. (d, left) 1 × 10⁵ Jeko-1 cells or (e, left) Mino cells were seeded in 24-well plate in triplicates and cultured under different conditions such as untreated (ctrl) or 20 nM cytarabine (CYT), 5 μg/ml of neutralizing anti-BAFFR antibody (anti-BAFFR), CYT+anti-BAFFR antibody, and CYT+ anti-BAFFF-R antibody+200 ng/ml rhBAFF. The cell number was measured for the time indicated, ****p < .0001 Jeko-1 or Mino cells treated with anti BAFF-R antibody as compared to combined treatment of cytarabine and anti-BAFFR antibody. *p < .05 Jeko-1 cells treated with cytarabine+anti-BAFFR antibody compared to cytarabine+anti-BAFFR antibody+recombinant BAFF-treated Jeko-1 cells. *p < .05 for Jeko-1 cells treated with cytarabine versus combined treatment of cytarabine and anti-BAFFR antibody. Error bars are from single experiment done with triplicate samples representative of three such experiments done with triplicates

Figure 2.

Modest knockdown of BAFF-R expression sensitizes MCL cells to cytarabine treatment in vitro. (a) Jeko-1 cells were infected with lentivirus shGFP, shBR3-3, or shBR3-4 for 72 hours. The transcriptional level of BAFF-R in Jeko-1 cells expressing shGFP, shBR3-3, or shBR3-4 was determined by RT-qPCR. ***p < .001 for Jeko-1 cells expressing shGFP compared to shBR3-3 and shBR3-3 compared to shBR3-4. (b) Graphical representation (on the left panel) showing the total population of early (Annexin V) and late apoptotic cells (PI) in one of three experiments with triplicate samples 96 hours after shGFP, shBR3-3, or shBR3-4 lentivirus infection in Jeko-1. The quantification data was shown on the right panel. ***p < .001 for Jeko-1 cells expressing shBR3-4 compared to shGFP; ns for Jeko-1 cells expressing shBR3-3 compared to shGFP. (c, left) 1 × 10⁵ Jeko-1 cells or Mino cells (d, left) expressing shGFP or shBR3-3 were seeded in 24-well plate in triplicates and the cell numbers were plotted. p > .05 is considered not significant (ns). Cell counts from (c, right) Jeko-1 shGFP and shBR3-3 cells or (d, right) Mino cells shGFP and shBR3-3 treated with cytarabine ***p < .001. One of the three experiments done with triplicate samples shown. (e, left) The apoptotic cell population was stained by Annexin V and PI followed by flow cytometry analysis Dot plot graph represents the total population of early and late apoptotic cells from (c). Apoptotic cell quantification data shown on the right panel. ***p < .001 for cells expressing shGFP and CYT treatment for 96 hours compared to cells expressing shGFP alone. ***p < .001 for cells expressing shBR3-3 compared to cells expressing shGFP after CYT treatment for 96 hours. Error bars are from single experiment done with triplicate samples representative of three such experiments done with triplicates. (f) Western blot analysis for the apoptotic marker of cleaved caspase-3 in Jeko-1 cells expressing shGFP or shBR3-3 after CYT treatment for 96 hours. GAPDH was used as a loading control

Figure 3.

BAFF stimulation failed to antagonize cytarabine-induced BAFF-R knockdown MCL cell death. (a) 1 × 10⁵ Jeko-1 cells expressing shGFP were seeded in 24-well plate in triplicates for a week. The cells were treated with different combination of components such as untreated (ctrl), CYT, rhBAFF, and CYT+rhBAFF. The cell number was plotted. ****p < .0001 represents untreated shGFP cells as compared to cytarabine-treated cells. *p < .05 represents cytarabine-treated shGFP cells in comparison to combined treatment of cytarabine and recombinant BAFF. Experiments shown are representative of three replicates from one experiment. (b) 1 × 10⁵ Jeko-1 cells expressing shBR3-3 were seeded in 24-well plate in triplicates for a week. The cells were treated with different combination of components such as ctrl, CYT, rhBAFF and CYT+rhBAFF. The cell number was plotted. ****p < .0001 represents untreated shBR3-3 cells as compared to cytarabine-treated cells. Experiments shown are one representative experiment of three experiments done with replicates; Not significant (ns) for the comparison of cytarabine-treated shBR3-3 cells in comparison to combined treatment of cytarabine and recombinant BAFF. (c) 1 × 10⁵ Mino cells expressing shGFP were seeded in 24-well plate in triplicates for a week. The cells were treated with different combination of components such as ctrl, CYT, rhBAFF, and CYT+rhBAFF. The cell number was plotted. ****p < .0001 represents untreated shGFP cells as compared to cytarabine-treated cells. ***p < .001 represents cytarabine-treated shGFP cells in comparison to combined treatment of cytarabine and recombinant BAFF. Experiments shown are representative of three replicates from one experiment. (d) 1 × 10⁵ Mino cells expressing shBR3-3 were seeded in 24-well plate in triplicates for a week. The cells were treated with different combination of components such as ctrl, CYT, rhBAFF, and anti-BAFFR. The cell number was plotted. ****p < .0001 represents untreated shBR3-3 cells as compared to cytarabine-treated cells; ns shows the comparison of cytarabine-treated shBR3-3 cells in comparison to combined treatment of cytarabine and recombinant BAFF. Error bars are from single experiment done with triplicate samples representative of three such experiments done with triplicates

Figure 4.

Modest knockdown BAFF-R expression significantly sensitizes the tumor to cytarabine treatment in vivo and prolongs survival of mice in the xenograft model. (a) Schematic representation of in vivo administration of cytarabine in Jeko-1 and Mino xenograft mice model. Jeko-1 and Mino cells were subcutaneously injected into NSG mice to generate solid tumor. When the tumor grew to measurable size, cytarabine was injected intraperitoneal for 3 consecutive days followed by 2 days’ interval gap and another 2 consecutive days’ injection as indicated. (b) Tumor volume was measured twice a week for two weeks and accordingly relative tumor growth rate was estimated for Jeko-1 xenograft cells. Left panel shows relative tumor growth rate of shGFP mice with shBR3-3. ****p < .0001 for relative tumor growth rate of mice with Jeko-1 cells expressing shBR3-3 and CYT treatment compared with shGFP and CYT treatment at different time points (right panel). N = 5. (c) Tumor volume was measured twice a week for two weeks and accordingly relative tumor growth rate was estimated for Mino xenograft cells. Left panel shows relative tumor growth rate of shGFP mice with shBR3-3. ****p < .0001 for relative tumor growth rate of mice with Mino cells expressing shBR3-3 and CYT treatment compared with shGFP and CYT treatment at different time points (right panel). N = 5. (d) Kaplan-Meier survival curve was graphed when the Jeko-1 cells tumor volume in mice reached 2000 mm3 and sacrificed the mice. ***p < .001 for survival time of mice with shBR3-3 and CYT treatment compared to mice with shGFP and CYT treatment. N = 5. (e) Kaplan-Meier survival curve was graphed when the Mino cells tumor volume in mice reached 2000 mm3 and sacrificed the mice. **<0.01 for survival time of mice with shBR3-3 and CYT treatment compared to mice with shGFP and CYT treatment. N = 5

Figure 5.

The binding of the VAY-736 to BAFF-R was competed by recombinant BAFF and anti-BAFF-R 11c1 antibody in MCL cell lines. (a) The binding of the VAY-736 antibody to JVM2 and Jeko-1 cells was detected by FITC anti-human IgG. Black histogram, control isotype; red histogram, VAY-736. (b) Inhibition of VAY-736 binding to the BAFF-R by rhBAFF in JVM2 cells as detected by FITC-anti-human IgG antibody. (c) VAY-736 binding to BAFF-R was competed by anti-BAFFR 11c1 in JVM2 cells. (d) Binding of VAY-736 to BAFFR was persistent in JVM2 as detected by FITC-anti-human IgG antibody (e) 1 × 10⁵ Jeko-1 cells were seeded in 24-well plate in triplicate and treated with CYT in the absence or presence of 10 μg/ml VAY-736 antibody. The cell number was measured for the time indicated. ***p < .001 for Jeko-1 cells with CYT and rhBAFF treatment compared with CYT alone on day 8. One of two experiments on triplicate samples was shown. (f) 4 × 10⁵/ml Jeko-1 cells were pre-incubated with PBS or VAY-736 for 72 hours and then stimulated with rhBAFF for 20 hours. Whole-cell lysates were made and western blot analysis was performed to detect protein levels of p52 and Pim2. GAPDH was used as the loading control

Figure 6.

VAY-736 enhances ADCC mediated by NK cells in vitro and in vivo. (a) Calcein-AM labeled JVM2 cells pre-incubated for 1 hour with control human IgG or VAY-736 were incubated with different ratio of NK cells (E:T) for 4 hours (b) Calcein-AM labeled Pt2 and Pt3 cells were pre-incubated 1 hour with control human IgG, VAY-736 followed by incubating with NK cells at (E:T of 5:1) for 4 hours. Statistical significance was calculated using unpaired student’s t-test. N = 3; **p < .01, ***p < .001. (c) Percentage of Jeko-1 cell lysis mediated by NK cells from 3 different normal donors. Cells were all pre-incubated with 10 ug/mL of VAY-736 by using E:T ratio of 5:1. Statistical significance was calculated using unpaired student’s t-test. N = 3; **p < .01, ***p < .001. (d) Percentage of JVM2 cell lysis mediated by NK cells from three different normal donors. Cells were all pre-incubated with 10 ug/mL of VAY-736 by using E:T ratio of 5:1. Statistical significance was calculated using unpaired student’s t-test. N = 3; **p < .01, ***p < .001. (e) Top, schematic depiction of Jeko-1 cells xenograft and VAY-736 treatment in mice. Bottom, relative tumor growth rate after different treatments. On the left panel, PBS group compare to VAY-736; On the middle panel, NK or NK+VAY-736 treatment compare to PBS control; on the right panel, NK/VAY-736 versus NK treatment. Statistical significance was calculated using two-way ANOVA. ****p < .0001 represents statistical significance between PBS versus NK+VAY-736 group, and NK in comparison to NK+VAY-736 group, ***p < .001 represents statistical significance between PBS versus NK group. N = 5 for left panel and N = 3 for middle and right panel. (f) Top, schematic depiction of JVM2 cells xenograft and VAY-736 treatment in mice. Bottom, relative tumor growth rate after different treatments. Statistical significance was calculated using two-way ANOVA. ***p < .0001 represents statistical significance between PBS versus NK+VAY-736 group, *p < .05 represents statistical significance between NK versus NK+VAY-736 group. N = 3