Evaluation of Bone Marrow in Fanconi Anemia Patients Treated with Briquilimab Antibody-Based Conditioning and TCRαβ⁺ T-Cell/CD19⁺ B-Cell Depleted Haploidentical Grafts

Background/Context: Fanconi Anemia (FA) is the most common cause of inherited bone marrow failure (BMF), with over 80% of patients developing symptoms by age 12. The hematologic aspects of the disease can be cured with allogeneic hematopoietic stem cell transplantation (allo-HSCT), though this has historically required the use of non-specific chemotherapy and/or irradiation to prepare the host bone marrow (BM), which causes systemic toxicities. We have recently launched a novel Phase 1b/2a clinical trial (NCT04784052) for FA patients in BMF using a targeted anti-CD117 monoclonal antibody, briquilimab, as a non-genotoxic method to facilitate the clearance of recipient hematopoietic stem and progenitor cells (HSPCs) from BM and thereby create BM niche space for donor HSPCs. This method replaces the conventional busulfan chemotherapy or total body irradiation that is often used in FA patients with non-sibling donors and is combined with immunosuppression followed by transplantation of TCRαβ⁺ T-cell and CD19⁺ B-cell depleted haploidentical grafts from healthy familial donors.

Aims & Methods: We aimed to understand the impact of this novel transplantation protocol on the BM and investigate the pathways involved in the survival and repopulation of HSPCs. As part of our corollary studies, we characterized BM HSPCs before, during, and after transplantation, assessing donor engraftment, BM DNA-repair efficiency, and effects on various BM cell types. We collected BM aspirates and biopsies at baseline, day 6 post-briquilimab administration, and at various time points post-HSCT (from 4 to 104 weeks). BM cell phenotyping and molecular profiling were conducted using flow cytometry, colony-forming assays, single-cell RNA sequencing (scRNA-seq), and targeted next-generation sequencing (NGS). These data were compared to healthy pediatric and adult controls (n = 3-10).

Interim Results: Our current patient cohort (n = 8) consists of patients aged 6 to 16 with FA of diverse FA genotypes (FANCA 75%, FANCC 12.5%, and FANCG 12.5%). At the present, 7 out of 8 patients are post-HSCT and each has exhibited rapid resolution of cytopenia (neutrophil engraftment by days 9-14, platelet engraftment by days 11-15) and high multi-lineage donor chimerism (total BM 98-100%, BM CD34⁺ 99-100% at last follow-up) with minimal toxicities. Two patients have reached the end of the study at 104 weeks post-HSCT both with 100% donor BM chimerism in CD34⁺ cells and all other lineages tested. BM cellularity was significantly increased in post-transplant patients who have reached 24 weeks (n=5, P= 0.0095) and 52 weeks (n=3, P= 0.0476) compared to their pre-transplant levels. We also observed correction of BMF with DNA-damage resistance in all seven patient BM cells challenged with 10 nM and 50 nM mitomycin C (MMC), and a significant increase in colony counts (P= 0.0238) in patients over 24-52 weeks post-HSCT. Additionally, post-HSCT hematopoiesis-related cytokines in the BM plasma, including EPO, KITLG, FLT3LG, ANGPT1, and CSF2, have shown a pattern of normalization to levels similar to healthy controls. At 24 weeks post-HSCT, transcriptomic analysis by scRNA-seq of HSPCs enriched from BM revealed a global reduction in p53, a regulator of inflammation, compared to baseline samples (n=2 paired samples sequenced to date). Further, BM HSC/MPP cells from these patients also displayed increased expression of hematopoiesis and HSC maintenance-related genes compared to healthy controls (n=6), with differentially expressed genes showing significant enrichment in translation and RNA processing. Importantly, no acquired mutations were detected in patient BM cells post-HSCT.

Conclusions & Next Steps: Our novel anti-CD117 antibody-based HSCT protocol appears to effectively replace the BM of FA patients with BMF, restoring healthy hematopoiesis and resistance to DNA damage. Notably, this was accomplished without the use of busulfan or total body irradiation, which is extremely toxic to FA patients. However, additional analyses and assessment of more samples with longer follow-up time points are needed and underway to further understand the full effects of this treatment on the BM microenvironment.

Bertaina:Gilead: Current Employment, Research Funding; Miltenyi: Current Employment, Honoraria; Neovii: Current Employment, Honoraria. Porteus:CRISPR Tx: Current equity holder in publicly-traded company; Allogene Tx: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Kamau Tx: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Czechowicz:Magenta Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Prime Medicine: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Jasper Therapeutics: Patents & Royalties, Research Funding; Global Blood Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months; Editas Medicine: Current equity holder in publicly-traded company, Patents & Royalties: Patent, no royalties; Beam Therapeutics: Current equity holder in publicly-traded company; Decibel Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: Patent, no royalties; Rocket Pharma: Research Funding; Spotlight Therapeutics: Consultancy, Current holder of stock options in a privately-held company; Teiko Bio: Current holder of stock options in a privately-held company; Inograft Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; STRM.Bio: Research Funding.