Post-transplant bendamustine reduces GvHD while preserving GvL in experimental haploidentical bone marrow transplantation

Abstract

Advances in haploidentical bone marrow transplantation (h-BMT) have drastically broadened the treatment options for patients requiring BMT. The possibility of significantly reducing the complications resulting from graft-versus-host disease (GvHD) with the administration of post-transplant cyclophosphamide (PT-CY) has substantially improved the efficacy and applicability of T cell-replete h-BMT. However, higher frequency of disease recurrence remains a major challenge in h-BMT with PT-CY. There is a critical need to identify novel strategies to prevent GvHD while sparing the graft-versus-leukaemia (GvL) effect in h-BMT. To this end, we evaluated the impact of bendamustine (BEN), given post-transplant, on GvHD and GvL using clinically relevant murine h-BMT models. We provide results indicating that post-transplant bendamustine (PT-BEN) alleviates GvHD, significantly improving survival, while preserving engraftment and GvL effects. We further document that PT-BEN can mitigate GvHD even in the absence of Treg. Our results also indicate that PT-BEN is less myelosuppressive than PT-CY, significantly increasing the number and proportion of CD11b(+) Gr-1(hi) cells, while decreasing lymphoid cells. In vitro we observed that BEN enhances the suppressive function of myeloid-derived suppressor cells (MDSCs) while impairing the proliferation of T- and B-cells. These results advocate for the consideration of PT-BEN as a new therapeutic platform for clinical implementation in h-BMT.

Keywords: bendamustine; bone marrow transplantation; cyclophosphamide; graft-versus-host disease; graft-versus-leukaemia.

Conflict of interest statement

The authors have declared that no conflict of interest exists.

© 2016 John Wiley & Sons Ltd.

Figures

Figure 1. Survival from GvHD following MAC and RIC haploidentical BMT (h-BMT) increases with PT-CY

CAF1/J mice (H-2d/a) received myeloablative conditioning (MAC) 1000 cGy or reduced intensity conditioning (RIC) 600 cGy total body irradiation (TBI) on day -1 and h-BM (107) with or without h-SC (3x107) from CB6F1 mice (H-2d/b) on day 0. Post-transplant cyclophosphamide (PT-CY) 75 mg/kg was given i.p. on days +3 and +4. Pooled data from 4 experiments (MAC; n=16) and 6 experiments (RIC; n=24). Kaplan-Meier survival curves were generated and analysed using the log-rank test. MAC:h-BMT/SC+PT-CY vs h-BMT/SC and h-BMT P<0.0001. RIC:h-BMT/SC+PT-CY vs h-BMT/SC P<0.0001 and vs h-BMT P<0.035.

Figure 2. PT-BEN preserves engraftment following MAC and RIC haploidentical BMT (h-BMT)

CAF1/J mice (H-2d/a) received myeloablative conditioning (MAC) or reduced intensity conditioning (RIC) total body irradiation (TBI), (A) h-BMT from CB6F1 mice (H-2d/b), (B) h-BMT/SC, (C) h-BMT/SC and post-transplant bendamustine (PT-BEN; 30 mg/kg day +3 or 15 mg/kg days +3 and +4 i.v.). Percentage donor chimerism determined by flow cytometry is shown. Peripheral blood cells were gated on live cells from FSC/SSC and then gated on H2kb+ cells. MAC n=16 mice (pooled data from 4 experiments), RIC n=16 (PT-BEN) or 32 (h-BMT and h-BMT/SC) mice/group (pooled data from at least 4 experiments).

Figure 3. PT-BEN is as efficient as PT-CY at mitigating GvHD in h-BMT

CAF1/J mice received myeloablative conditioning (MAC) or reduced intensity conditioning (RIC) total body irradiation (TBI) on day -1 and h-BM (107) with h-SC (3x107) from CB6F1 mice on day 0. Post-transplant bendamustine (PT-BEN) 30 mg/kg was given on day +3 and post-transplant cyclophosphamide (PT-CY) 75 mg/kg was given on days +3 and +4. Kaplan-Meier survival curves were generated and analysed using the log-rank test. (A) Overall survival is shown. n=12-16 mice/group, pooled data from at least 3 experiments. MAC:h-BMT/SC vs h-BMT/SC+PT-BEN and vs h-BMT/SC+PT-CY P<0.0001, h-BMT/SC+PT-BEN vs h-BMT/SC+PT-CY P=ns.RIC:h-BMT/SC vs h-BMT/SC+PT-BEN P<0.004 and vs h-BMT/SC+PT-CY P=0.017. h-BMT/SC+PT-BEN vs h-BMT/SC+PT-CY P=ns. (B) Mean per cent weight change with SEM based on pre-transplant weight. (C) Mean clinical GvHD scores (based on weight, skin integrity, fur, activity and posture) with SEM. Shown are representative data from 4 experiments. n=4 (RIC) or 8 (MAC) mice/group.

Figure 4. PT-BEN and PT-CY show comparable levels of colonic and hepatic GvHD

CAF1/J mice received reduced intensity conditioning total body irradiation on day -1 and h-BM (107) with h-SC (3x107) from CB6F1 mice on day 0. Post-transplant bendamustine (PT-BEN) 30 mg/kg and post-transplant cyclophosphamide (PT-CY) 75 mg/kg were given on days +3 and +4. (A) Proximal and distal colons were collected and blindly scored for abnormalities. n=4 mice/group. Total colon scores (sum of proximal and distal scores) from days +21, +35 and +49 are shown. (B) Representative images of haematoxylin and eosin staining of distal colons collected on day +21 are shown at 50X magnification. (C) Representative images of haematoxylin and eosin staining of livers collected on day +21 are shown at 50X magnification.

Figure 5. PT-BEN is protective against GvHD even in the absence of donor CD25+ T-cells

Recipient CAF1/J mice received reduced intensity conditioning and T-cell depleted h-BM (107) with 6x106 total T-cells or 6x106 CD25-depleted T-cells from CB6F1 mice on day 0. Post-transplant bendamustine (PT-BEN) 30 mg/kg was given on day +3. Shown are survival data and mean clinical graft-versus-host disease scores with SEM. Representivative data from 2 experiments, n=6 mice/group. Kaplan-Meier survival curves were generated and analysed using the log-rank test. CD25- T vs CD25- T+PT-BEN P<0.002, total T vs CD25- T+PT-BEN P=ns, total T vs CD25- T P<0.03.

Figure 6. PT-BEN is less myelosuppressive than PT-CY following MAC and RIC h-BMT

CAF1/J mice received myeloablative conditioning (MAC) or reduced intensity conditioning (RIC) total body irradiation (TBI) on day -1 and h-BMT/SC from CB6F1 mice on day 0. Post-transplant bendamustine (PT-BEN) 30 mg/kg was given on day +3 and post-transplant cyclophosphamide (PT-CY) 75 mg/kg was given on days +3 and +4. Peripheral blood was collected on the indicated days by tail tipping. (A) Day +7 white blood cell (WBC) counts broken down into myeloid/lymphoid differentials are shown with SEM. Blood was analysed using a HemaVet blood counter and broken down into neutrophils, monocytes, eosinophils, basophils (myeloid) and lymphocytes (lymphoid). MAC n=8 mice/group, RIC n=14 mice/group (pooled data from 2-3 experiments). (B) Absolute numbers of donor CD4+FoxP3+ Treg are shown. n=4-8 (pooled data from 2-3 experiments). (C, D) Absolute numbers of donor CD4, CD8 and B-cells in peripheral blood are shown. Cells were stained with anti-H2kb, CD4, CD8 and B220 and analysed by flow cytometry. Cells were gated on live cells from FSC/SSC, H2kb+ cells and then on CD4+, CD8+ or B220+ and absolute cell number was calculated based on total WBC count. (E) Absolute number of donor CD11b+Gr-1hi cells is shown. Day +7 and +14 blood was stained with anti-H2kb, Gr-1 and CD11b and analysed by flow cytometry. Cells were gated on live cells from FSC/SSC, H2kb+ cells and then on CD11b+Gr-1hi. Gating strategy and representative flow from RIC day +14 are shown. Absolute cell number was calculated using the WBC count and difference were analysed using Mann-Whitney test. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.

Figure 7. BEN treatment in vitro enhances MDSC function and decreases the proliferation of CD4+ and CD8+ T-cells and B-cells

(A) Myeloid-derived suppressor cells (MDSCs) were generated using bone marrow (BM) cells from naïve CB6F1 mice. BM cells were cultured for 3 days with 10 ng/ml murine IL6, murine GM-CSF and human G-CSF and various concentrations of bendamustine (BEN; 0-100 μM). MDSCs were then co-cultured for 3 days with CellTrace Violet stained, stimulated total T-cells from naïve CB6F1 spleens at a ratio of 1 MDSC to 2 T-cells. Average % T-cell proliferation (with SEM) after 3 days of co-culture is shown. Pooled data from 5 experiments and representative ModFit analysis with proliferation index are shown. This includes T-cells without MDSCs as a negative control for suppression. Significance is shown as compared to 0 μM BEN, using Mann-Whitney tests. (B) T-cells isolated from the spleens of naïve CB6F1 mice were CellTrace stained, stimulated with CD3/CD28 beads and cultured for 3 days in the presence of various concentrations of BEN. On day 3, viability was determined and T-cells were stained with anti-CD4 and CD8 for proliferation analysis by flow cytometry. Cells were gated on live cells, CD4+ or CD8+ and then CellTrace fluorescence was analysed. Shown are pooled data from 4 experiments. (C) B-cells isolated from the spleens of naïve CB6F1 mice were CellTrace stained, stimulated with 10 μg/ml lipopolysaccharide and cultured for 5 days in the presence of various concentrations of BEN. Shown are pooled data from 4 experiments. Viability and average percent proliferation as compared to the culture with 0 μM BEN are shown, using Mann-Whitney tests. *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001.

Figure 8. BEN has minimal direct anti-tumour activity against A20 leukaemia and preserves GvL when given post-h-BMT

A20 leukaemia-bearing (A) BALB/c and (B) NOD-scid IL2Rγnull were treated with bendamustine (BEN) 30 mg/kg or cyclophosphamide (CY) 75 mg/kg 5 and 6 days post-injection of 106 A20 cells. CY vs BEN P<0.002 in BALB/c and P<0.006 in NOD-scid IL2Rγnull, n=4 (BALB/c) or 8 (NOD-scid) mice/group. (C) CAF1/J mice received 106 A20 tumor cells i.v. on day -4, RIC TBI on day -1 and h-BM (107) or h-BMT with h-SC (3x107) from CB6F1 mice on day 0. PT-BEN 30 mg/kg as a single dose or divided over 2 days was given starting on day +3. Pooled data from 5 experiments, n=20-34 mice/group. Kaplan-Meier survival curves were generated and analysed using the log-rank test. h-BMT vs h-BMT/SC+PT-BEN P<0.002. (D) CAF1/J mice received RIC TBI on day -1 and h-BMT or h-BMT/SC from CB6F1 mice on day 0. PT-BEN (33 mg/kg) or CY (150 mg/kg) was given on day +3 and 1x105 A20-Luc cells were injected i.v on day +4. h-BMT/SC+PT-BEN vs h-BMT P<0.0004 and vs h-BMT/SC+PT-CY P<0.0005, h-BMT/SC+PT-CY vs h-BMT P=ns. Pooled data from 2 experiments, n=8 mice/group.

Figure 9. PT-BEN preserves GvL when given post-h-BMT

(A) CAF1/J recipient mice received reduced intensity conditioning on day -1 and T-cell depleted h-BMT or h-BMT/SC from CB6F1 mice on day 0. Post-transplant bendamustine (PT-BEN) or post-transplant cyclophosphamide (PT-CY) was given day +3 and 0.5x106 A20-Luc cells on day +4. Mice were injected with luciferin and imaged serially. At times, cages were imaged concurrently, so images are cropped to only show mice from the indicated group. (B) Quantification of (A) is shown in ln(photons/s/mouse). Group average with SEM is shown (left) until any mouse from the group died and analysed using Mann-Whitney tests. Individual mouse values are also shown (right). Images were analysed using AmiView. n=4-5 mice/group.