Irradiation- and busulfan-free stem cell transplantation in Fanconi anemia using an anti-CD117 antibody: a phase 1b trial

Abstract

Current hematopoietic stem cell transplantation (HSCT) conditioning strategies cause widespread tissue damage and systemic toxicities, especially in patients with DNA-repair deficiencies such as Fanconi anemia (FA). We have developed an alternative conditioning approach that incorporates the anti-CD117 antibody, briquilimab, which targets host hematopoietic stem and progenitor cells in place of genotoxic irradiation- and busulfan-based chemotherapy. Here we report a phase 1b clinical trial in patients with FA and bone marrow failure, evaluating safety and efficacy of briquilimab-based conditioning in combination with rabbit anti-thymocyte globulin, cyclophosphamide, fludarabine and rituximab immunosuppression and T cell receptor (TCR)αβ⁺ T cell-depleted and CD19⁺ B cell-depleted haploidentical HSCT. Primary endpoints of the trial included safety and engraftment, and secondary endpoints included pharmacokinetic measures and hematological and immunological recovery. All three patients have each undergone 2 years of follow-up to complete the phase 1b analysis. No treatment-emergent adverse events or acute graft-versus-host disease was observed. Patients experienced minimal toxicities, with typical mucositis and no veno-occlusive disease. Median neutrophil engraftment was 11 days (range 11-13 days) with robust donor chimerism up to 2 years post-HSCT (99-100%), meeting the primary endpoints of the study. Briquilimab cleared in each patient before HSCT without the need for adjustment. Red blood cell, platelet and lymphocyte recovery was comparable to previous reports with TCRαβ⁺ T cell-depleted and CD19⁺ B cell-depleted grafts. All patients are alive and well with resolution of earlier chromosomal breakage abnormalities in peripheral blood lymphocytes post treatment. These data demonstrate the broad potential of this protocol in maintaining HSCT efficacy while reducing toxicity. The phase 2 trial is ongoing (ClinicalTrials.gov identifier: NCT04784052 ).

Fig. 1. Overview of study investigating transplantation of αβ-depleted HSPC grafts with irradiation- and busulfan-free, antibody-containing, reduced-intensity conditioning in people with FA.

a, CONSORT diagram displaying all three patients who were enrolled and screened, with each undergoing conditioning, transplantation and 2 years of follow-up (f/u) on the phase 1b study. b, Schematic displaying treatment utilized with a reduced-intensity conditioning regimen that was irradiation- and busulfan-free, consisting of the anti-CD117 monoclonal antibody (mAb) briquilimab, rATG, fludarabine, cyclophosphamide and rituximab. The graft used for HSCT was obtained from the peripheral blood of a haploidentical family member after filgrastim and plerixafor mobilization and apheresis, and was subsequently infused after depletion of TCRαβ⁺ T cells and CD19⁺ B cells. All three patients have fully completed the phase 1b study with 104 weeks of follow-up. Schematic in b created using BioRender (https://biorender.com). ^aATG and fludarabine immunosuppression drug monitoring. ^bOnly given if <40 CD34⁺ cells µl⁻¹ in peripheral blood. ^cATG dosing was adjusted between patients: patient 1 at 2.5 mg kg⁻¹ day −9 to −7, all others 4 mg kg⁻¹ day −10 to −8.

Fig. 2. Briquilimab antibody clearance to enable transplantation of donor HSPCs.

Predictable briquilimab clearance could support successful HSCT at the intended day of transplantation in all three treated patients. In addition, the briquilimab level was less than the threshold of <1,500 ng ml⁻¹ on the day of stem cell infusion in all patients.

Fig. 3. Patient engraftment after briquilimab-containing conditioning and allo-HSCT.

a–c, All three patients promptly engrafted neutrophils (a), platelets (b) and RBCs (c) within 2 weeks of HSCT with resolution of earlier cytopenia. d,e, Near-complete multilineage donor chimerism at both short- and long-term timepoints post-HSCT in peripheral blood (PB) (d) and bone marrow (BM) (e). Data are presented as mean ± s.e.m. Each data point represents the average of measurements from three patients.

Fig. 4. Immune recovery after briquilimab-containing conditioning and allo-HSCT.

a, Efficient immune recovery of CD3⁺ T cells, CD19⁺ B cells and CD16⁺CD56⁺ NK cells post allo-HSCT, comparable to other T cell-depleted HSCT approaches. Data are presented as mean ± s.e.m. Each data point represents the average of measurements from three patients. b, Peripheral blood chromosomal breakage studies using the clastogenic agent DEB showing FA-related chromosome breakage in lymphocytes from all patients at screening. Chromosomal breakage was normalized in lymphocytes in each of the patients post-HSCT, with no notable breakage, resembling control values from unrelated healthy donors. Data are presented as mean values, with each point representing the average from 50 cells.

Fig. 5. Reassuring patient course after briquilimab-containing conditioning and allo-HSCT.

All three patients were treated without developing any investigational product severe adverse events. a, All patients remain engrafted with 100% event-free survival (EFS) and no primary or secondary graft failure, which is most typically observed within 6 months of HSCT despite the use of haploidentical, mobilized, peripheral blood donor grafts. b, No acute GvHD observed in any patient. c, Chronic GvHD observed in only one patient.

Extended Data Fig. 1. Study Goals and Approaches Used.

Three complimentary goals of avoiding DNA-damage to cells from individuals with Fanconi Anemia, optimizing hematopoietic grafts and close pharmacokinetic monitoring were incorporated in this clinical trial.

Extended Data Fig. 2. Clearance of rATG and Fludarabine in Context of Conditioning Regimen Containing Briquilimab.

Given the graft rejection that was observed in prior dose de-escalation of TBI from FA allo-HSCT protocols, close pharmacokinetic monitoring of rATG and fludarabine was performed to ensure concurrent optimal immunosuppression was administered. a) rATG: Patient 1 received 2.5 mg/kg IV for 3 doses starting on day −9. The pre-HSCT AUC was estimated to be below goal at 14.6 AU*day/mL, while post-HSCT AUC was estimated to be within goal at 2.31 AU*day/mL. Given the low pre-HSCT AUC for Patient 1, the rATG dose was increased for Patients 2 and 3 to 4 mg/kg IV for 3 doses starting on day −10. b) Fludarabine: Cumulative AUC (cAUC) for Patient 1 was within the previously published goal and the same dosing of 35 mg/m² IV for 4 doses was maintained for Patients 2 and 3.

Extended Data Fig. 3. Blood Count at Screening and Recovery Post αβdepleted-HSCT.

All patients were monitored post-HSCT with donor engraftment that enabled rapid tri-lineage blood count recovery in each patient (n = 3). Patient 2 developed immune mediated hemolytic anemia and thrombocytopenia after HSCT that resolved post treatment.

Extended Data Fig. 4. Immune Subsets at Screening and Recovery Post αβdepleted-HSCT.

All patients were monitored post-HSCT with immune recovery similar to previous reports with this type of donor graft (n = 3). Decreased CD19⁺ B-cells occurred in patient 2 at 24 weeks post-HSCT due to treatment for immune mediated hemolytic anemia and thrombocytopenia.

Extended Data Fig. 5. Bone Marrow Hematopoiesis at Screening and Recovery with Restored Hematopoiesis and DNA-Damage Resistance Post αβdepleted-HSCT.

a) Minimal in vitro colony forming counts (CFCs) at screening (red) indicative of reduced HSPC activity due to BMF in each individual with FA pre-treatment compared to healthy adult and pediatric controls (black) with increased CFC activity post allo-HSCT in all patients assessed (grey, gold and teal). Data are presented as mean ± SEM. Each bar represents an independent sample, with values averaged from three technical replicates. b) No mitomycin C (MMC)-resistance was observed in BM cells on research testing at patient enrollment in each of the treated individuals, indicative of FA disease with no somatic mosaicism at screening that subsequently increased post-HSCT with normalized 10 nM and 50 nM MMC-resistance in all patients due to engraftment of donor HSPCs without FA defects. Data are presented as mean ± SEM. Each data point represents an independent sample, with values averaged from three technical replicates. Parametric unpaired t-test (two-tailed) was used for statistical analysis.