CD45-Directed Radioimmunotherapy with the Alpha-Emitter Astatine-211 As Conditioning for Autologous Hematopoietic Stem/Progenitor Cell Gene Therapy: Results from a Nonhuman Primate Model

Background: Ex vivo gene-modified autologous cell products are increasingly explored to cure monogenic disorders (e.g. sickle cell disease, thalassemia). Efficient removal of bone marrow (BM)-resident hematopoietic stem/progenitor cells (HSPCs) is a requirement for successful engraftment of such cell products. To accomplish this, various conditioning regimens have been utilized, each with their own unique profile of target specificity and on- and off-target toxicities. Currently most widely used is busulfan but unwanted non-hematologic toxicities have provided the impetus to develop more specific approaches that spare normal tissues better. Monoclonal antibodies (mAbs) conjugated with either small molecule toxins or radionuclides to selectively target phenotypically distinct cells have gained attention for this purpose. Of particular interest as target is CD45, a glycoprotein expressed at very high copy number exclusively on almost all blood cells. CD45-targeted radioimmunotherapy (RIT) with the beta-emitter, iodine-131, has proven effective to augment conditioning before allogeneic hematopoietic cell transplantation (HCT). Here, we evaluated CD45-directed RIT using a the highly potent alpha emitter, astatine-211 (²¹¹At) as payload as sole conditioning agent before autologous HCT of ex vivo gene-edited HSPCs in a nonhuman primate (NHP) HCT and gene therapy model.

Methods: A humanized version of the human/NHP cross-reactive CD45 mAb, BC8 (HuBC8), was conjugated with isothiocyanatophenethyl-ureido-closo-decaborate(2-) (HuBC8-B10), a boron cage molecule to enable subsequent labeling with ²¹¹At as done in our early phase clinical trials. Simultaneously, NHP CD34⁺ cells were mobilized with G-CSF/AMD3100, collected via leukapheresis, and cryopreserved after being gene modified ex vivo using adenine base editors to reactivate fetal hemoglobin (HBG) production as well as to delete CD33. 72 hours after administration of a single dose of ²¹¹At-labeled HuBC8-B10, gene-edited CD34⁺ cells were thawed and infused. Animals received 300 µCi/kg (n=2) or 400 µCi/kg (n=1) of ²¹¹At with 0.5 mg/kg of HuBC8-B10. All animals were monitored for toxicities, and blood count recovery as well as engraftment of gene-modified HSPCs and blood lineages which were assessed serially using flow cytometry and next generation sequencing.

Results: The editing efficiency of CD33 and HBG in the infusion product ranged from 50-77% and 15-38%, respectively, with no measurable impact on cell viability or erythro-myeloid differentiation potential of edited cells in colony-forming cell assays. A total of 2-4x10⁶ CD34⁺ cells/kg were infused into the animals and rapid recovery of neutrophils and platelets seen in between 6-8 and 10-13 days, respectively. Transient weight loss over the first 20-30 days was seen (n=3) and gastric ulcer treatment needed (n=1). An ²¹¹At dose of 300 µCi/kg led to incomplete myeloablation with neutrophils remaining above 400/µl, whereas full myeloablation was seen at a dose of 400 µCi/kg. Ablation of monocytes, lymphocytes, and platelets was seen in all animals, whereas CD45-negative erythrocytes were spared and the hemoglobin remaining stable throughout the study. Two animals were entirely transfusion-independent and the third animal receive a single platelet transfusion. Dose-dependent engraftment of gene-editing in the peripheral blood was seen with 20-40% CD33-negative cells and 5-10% HBF reactivation in the two animals receiving ²¹¹At at 300 µCi/kg as compared to 80% of CD33-negative cells and 20% HBF reactivation in the single animal receiving ²¹¹At at 400 µCi/kg. Full recovery of the BM stem cell compartment was confirmed at 3-month post-transplant by flow cytometry.

Conclusion: CD45-targeted alpha emitter-based RIT with ²¹¹At enables stable engraftment of ex vivo gene-edited autologous stem cell products. Our studies identify ²¹¹At-CD45 RIT as a targeted alternative for myeloablative conditioning followed by autologous transplantation of gene-modified HSPCs. ²¹¹At-HuBC8-B10 is well tolerated with only minimal adverse reactions observed in NHPs. While ²¹¹At-CD45 RIT demonstrates selective depletion of CD45-positive white blood cells as well as HSPCs in the BM, CD45-negative erythrocytes are spared, likely contributing to the minimal supportive care needs needed following autografting.