Introduction: Standard chemotherapy and irradiation-based conditioning for hematopoietic stem cell transplant (HCT) and autologous-transplant-based gene therapy (GT) is associated with significant toxicities. Because of this, there is a concerted effort to develop targeted conditioning approaches, including antibody-drug conjugates (ADCs). Depending on their specificities, ADCs can selectively deplete hematopoietic stem and progenitor cells (HSPCs). CD117, a receptor primarily (but not exclusively) expressed on HSPCs, represents a natural ADC target. However, previous attempts at creating a safe and effective CD117-ADC have been hampered by Grade 5 SAEs related to the CD117 antibodies themselves (mast cell degranulation), and to toxicities possibly related to the drug payload.

ADC-Design:

To address these issues, we developed a novel CD117-ADC (YTD005), with 4 key design features: 1st, YTD005 comprises an antagonistic anti-CD117 clone, discovered as a high affinity binder by phage display panning. 2nd, it comprises a monomeric antibody fragment that lacks Fc-effector functions, thereby substantially reducing mast cell degranulation. 3rd, the Fab' design enables rapid systemic elimination, which limits depletion of infused stem cells. 4th,YTD005 incorporates the tubulin inhibitor payload MC-MMAF at a theoretical DAR of 4. MC-MMAF is a non-cell-permeable toxin with low bystander effect, and incorporates a non-cleavable linker between MMAF and the CD117 Fab', further reducing off-target payload effects. Here, we report safety and engraftment outcomes with YTD005 in a NHP model of GT using CD34+ cells transduced with BCH-BB694, a lentiviral vector targeting BCL11A.

Methods: Rhesus Macaques (RM) underwent a GCSF-mobilized bone marrow harvest for manufacturing the BCH-BB964-transduced product. Based on extensive dose-finding studies in mice (using a tool ADC), cynomolgus macaques and RM, recipients received 1.5mg/kg/day YTD005 as a continuous infusion from Day -9 - -2. After 2 rest days (Day -2, -1) for ADC clearance, the GT product was infused. Animals were monitored for up to 1-year post-GT for toxicity and engraftment, with primary engraftment data obtained at Day +30.

Results: 3 RMs underwent autologous GT following YTD005 conditioning. YTD005 achieved robust depletion of Lin- CD34+ HSPCs, with a median depletion of 98.2% (range, 97.8-99.6%), as well as of Lin-CD34+CD90+CD45RA- HSPCs (median=96.8%, range 94-99.6%). The regimen was well tolerated, with no significant non-hematologic toxicities, including no hepatic, renal, cardiac, or pulmonary complications, and no evidence for mast cell degranulation. Recipients received a median of 5.7×10⁶ CD34+ cells/kg. The mean vector copy number (VCN) in the product was 4.2 copies per diploid genome (c/dg, range 3.6-4.6). All animals developed transient pancytopenia. Neutrophils reached a median nadir of 100/mL (range 100-300) at a median of day +9 (range, day +7 - +11), with engraftment occurring by a median of day +13 (range, +13 - +14). Platelets reached a median nadir of 56K (range 43-102K) at a median of day +3 (range, –4 - +7) followed by engraftment by median day +13 (range, +7 - +14).

Engraftment was assessed via peripheral blood (PB) VCN (c/dg) and the % of colonies derived from CD34+ bone marrow cells with a VCN >1. The primary outcome was % engraftment at Day +30, with all 3 animals reaching this timepoint. At 30 days, the mean blood VCN was 0.082 +/- 0.028 (indicating ~8% of PB contained the transgene), and the mean % colonies with a VCN >1 was 15.6% (range, 13.4-20%). 2 animals have been followed for >3 months, with mean 3-month blood VCN = 0.087 (~8% of cells), and the % colonies with a VCN >1 = 15.8% (range, 15.7-15.9%). One animal has thus far completed 1-yr follow up (other animals ongoing), and demonstrates stable engraftment (PB VCN = 0.1 (~10% of PB cells), and the % colonies with a VCN >1 was 15.4%).

Conclusions: We designed a novel CD117-ADC with 4 key features (antagonistic epitope, lack of Fc function, short half-life, and non-cell-permeable payload) to optimize both safety and efficacy. We demonstrate that a single infusional treatment with this ADC leads to engraftment of gene-modified cells, with mixed chimerism that was stable up to >1 year post-infusion.

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2025
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