Phase II Study Evaluating the Safety and Efficacy of BL-8040 for the Mobilization of Donor Hematopoietic Stem and Progenitor Cells for Allogeneic Hematopoietic Cell Transplantation and Phenotypic Characterization of the Leukapheresis Product

Background. Mobilized hematopoietic stem/progenitor cells (HSPCs) are the most commonly used graft source for hematopoietic cell transplantation (HCT). Current practices for harvesting HSPCs with G-CSF and/or plerixafor are costly, involve a multi-day procedure with suboptimal HSPC yields in up to 30% of patients and are associated with some morbidity and mortality. Here, we sought to test BL-8040, a high affinity CXCR4 antagonist with rapid mobilizing kinetics and long receptor occupancy, in donors for allogeneic HCT (alloHCT).

Methods. The primary endpoint was to assess the efficacy of a single injection of BL-8040 to mobilize ≥ 2 x 10⁶ CD34⁺ cells/kg of recipient weight after no more than two leukapheresis (LP) collections. Donors received BL-8040 by subcutaneous (SC) injection 3 hours prior to LP on day 1. The study was conducted in 2 parts. Part 1 enrolled HLA-identical pairs and 14 donors were treated with 1 mg/kg BL-8040. Part 2 enrolled HLA-identical and haploidentical pairs and treated 11 donors with 1.25 mg/kg BL-8040. When < 5x10⁶ CD34+ cells/kg recipient weight were collected, a second injection of BL-8040 and LP collection was performed the next day. Immunophenotyping of lymphoid, myeloid, and HSPC subsets in the blood and LPs was performed by flow cytometry.

Results. 25 donor-recipient pairs were enrolled in the study. The median age of donors was 55 years (range 20-69); 18 were fully HLA-matched siblings, and 7 were haploidentical donors. One donor was replaced per protocol because of problems with vascular access. All 11 donors treated at the 1.25 mg/kg dose and 22/24 (92%) patients overall collected the minimum goal of 2 x 10⁶ CD34+ cells/kg recipient weight in 2 LPs. 9/11 donors treated with 1.25 mg/kg BL-8040 collected the target of 5 x 10⁶ CD34+ cells/kg recipient weight. The median CD34+ cell count was 15 cells/µL blood at 3-4 hours after BL-8040 and was maintained for > 24 hours. BL-8040-adverse related events consisted primarily of grade 1-2 injection site reactions and transient systemic reactions (hives). Recipients were a median of 58 years of age (26-71) with an ECOG PS of 0-2 undergoing alloHCT for AML (n=16, 64%), ALL (n=4, 16%), MDS (n=3, 12%), MPN (n=1, 4%) or Hodgkin lymphoma (n=1, 4%). 22 recipients were transplanted with BL-8040-mobilized grafts. Time to neutrophil engraftment occurred at a median of 13 days (n=22, range 11-26) and platelet engraftment occurred at a median of 20 days (n=20, range 15-41; no platelet nadir for 1 pt and no engraftment in 1 pt). With a median follow-up of 258 days, 5 deaths have been observed on study. These deaths were due to sepsis (Day +84), complications of sepsis in setting of grade 3 GVHD (Day+67), AML relapse (Day+143,+166), and complications of intrabdominal infection (Day+252). Grade II acute GVHD was observed in 2/22 recipients (9%) both of which resolved. Grade III-IV GVHD was observed in 5 out of 22 recipients (23%), of which 3 resolved. Flow cytometric evaluation of HSPCs purified from LPs by CD34 immunoselection revealed that BL-8040 preferentially mobilized CD34+ plasmacytoid dendritic cell (pDC) precursors (pre-pDCs) that express high levels of CXCR4 (Fig. 1A). These pre-pDCs are the immediate progenitors of pDCs; a distinct DC population capable of producing large amounts of type 1 interferons in response to viral infections. In contrast to plerixafor, BL-8040 remained bound (as determined by inhibition of anti-CXCR4 binding) to CXCR4 on all lymphoid, myeloid, and HSPCs after LP (Fig. 1B and data not shown). BL-8040 induced pan-mobilization of all major myeloid and lymphoid subsets, with maximum relative changes in the absolute numbers of circulating pDCs, B cells, basophils, myeloid DCs (mDCs), CD8+ T cells and CD14+HLA-DR+CD16lo classical monocytes (Fig. 1C). In general, the magnitude of mobilization of each subset correlated with the level of surface CXCR4 at baseline (Fig 1C, gray bars, R² = 0.4; P = 0.02). Although there was pan mobilization of all CD8+ and CD4+ T cell subsets, BL-8040 preferentially mobilized naïve and central memory CD8⁺ T cells relative to the other T cell subsets (Fig. 1D).

Conclusions. Treatment of normal donors with a single dose of BL-8040 results in rapid and sustained mobilization of HSPCs for use in allogeneic transplant. BL-8040 mobilized grafts result in rapid and reliable engraftment in transplant recipients. Additional data is required to assess the impact of BL-8040-mobilized grafts on rates of GVHD and relapse.

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