Anti-CD117 Plus Anti-Sirpα or Fc-Silenced Anti-CD47 Enables Hematopoietic Stem Cell Depletion and Syngeneic Engraftment without Development of Severe Anemia

INTRODUCTION: In preparation for hematopoietic stem cell transplantation (HSCT), recipients are conditioned with chemotherapy and irradiation to enable donor hematopoietic stem cell (HSC) engraftment. The acute and chronic toxicities caused by these conditioning regimens negatively impact quality of life, life expectancy, and fertility, and may prevent unfit patients from undergoing HSCT at all. Antibody-drug conjugates (ADC) have been shown to enable HSCT within and across immunological barriers in preclinical models with fewer adverse effects (Persaud et al (2021); J Clin Invest), but even ADCs have toxicity risks caused by systemic exposure to the highly potent compounds used as ADC payloads. By avoiding exposure to cytotoxic and genotoxic agents altogether, naked antibody-based conditioning regimens would have the optimal long-term safety profile. Safer, less intense conditioning regimens would be especially attractive for HSCT used for cure of non-malignant hematologic diseases like sickle cell disease (SCD), in which the conditioning regimen does not need to provide antitumor benefit and induction of stable mixed chimerism is sufficient for cure. We have recently explored naked antibody targeting of CD117 (cKit) and CD47 for allogeneic HSCT (Persaud et al (2024), Blood Adv), but this regimen induces a profound peritransplant anemia which would make it particularly undesirable for HSCT conditioning in SCD recipients who are already anemic at baseline. As the Fc-region is required for red blood cell (RBC) destruction induced by anti-CD47, we hypothesized that an Fc-silenced anti-CD47 would enable HSC depletion when combined with anti-cKit without inducing anemia. Moreover, the RBC toxicities induced by anti-CD47 could also be avoided by instead using an anti-SIRPα antibody to block CD47-SIRPα signaling.

METHODS: A recombinant, Fc-silent version of anti-CD47 clone mIAP301 (Gilead Sciences; mouse IgG2a isotype) was generated by introducing LALA mutations (L234A, L235A) to the Fc region. To condition mice for HSCT with anti-CD47 and anti-cKit, a 100 μg priming dose of anti-CD47 was given 8 days before HSCT (d-8), followed by 500 μg anti-cKit (clone ACK2; BioLegend) on d-6 and five daily 500 μg doses of anti-CD47 (d-6 to d-2). For blockade of SIRPα, 500 μg anti-SIRPα (clone P84; BioLegend) was given on d-6 or on d-6 plus d-5 in lieu of anti-CD47 treatment. Bone marrow harvested on d0 was analyzed for HSC depletion by flow cytometry (CD150⁺CD48^-Lineage^-Sca1⁺cKit⁺ cells) and by colony formation assay in methylcellulose culture. Spleens and peripheral blood collected on d0 were analyzed for effects on mature immune cell subsets and complete blood counts (CBC). For syngeneic HSCT studies, B6 recipients were transplanted with 10 x 10⁶ B6-GFP whole bone marrow cells; CBCs and peripheral blood donor chimerism (GFP⁺) were analyzed monthly.

RESULTS: As has been previously reported, anti-CD47/cKit conditioning using anti-CD47 clone mIAP410 effectively depleted the HSC niche but induced a transient but severe anemia (hematocrit = 9.1%; lower reference limit for murine hematocrit = 35.1%) leading to significant morbidity. This anemia was ameliorated without impairing HSC depletion by either using an Fc-silenced anti-CD47 antibody (hematocrit = 28.9%) or by targeting SIRPα (hematocrit = 29.6%) Notably, targeting SIRPα avoided the prominent antigen sink effects observed in targeting the ubiquitously expressed CD47, requiring only a single anti-SIRPα dose given alongside anti-cKit on d-6 to achieve HSC depletion (versus five full doses plus a priming dose of anti-CD47). Finally, preliminary HSCT studies with anti-cKit/SIRPα targeting shows that this regimen enables syngeneic engraftment with ~50% myeloid lineage donor chimerism. Importantly, no morbidity or mortality was observed in mice conditioned with these protocols.

CONCLUSIONS: Combining anti-SIRPα or Fc-silent anti-CD47 with anti-cKit yielded two novel HSCT conditioning strategies that target the CD47/SIRPα axis without inducing severe RBC toxicity, greatly improving the safety of this radiation, chemotherapy, and toxic ADC payload-free approach. Studies are currently underway to evaluate these conditioning regimens in the Townes SCD model, using syngeneic marrow from wild-type HbAA-expressing littermates and autologous gene-corrected HSCs from HbSS mice as donor graft sources.