Engineering an antibody-discernible and functional CD45 variant on hematopoietic stem and progenitor cells via base editing

Hematopoietic stem cell transplantation (HCT) stands as the unique curative recourse for various hematological malignancies. However, the current standard-of-care relies on untargeted toxic conditioning regimens to deplete malignant cells and remove cells from host’s bone niches. These untargeted regimens limit HCT’s widespread use due to severe off-target toxicity effects. To counteract this roadblock, antigen-specific cell-depleting agents have emerged as promising targeted alternatives. Pairing antigen-specific conditioning regimens with transplantation of depleter-shielded HSPCs has shown promise in the treatment of malignancies characterized by the overexpression of specific cell-surface markers (e.g. CD123 or CD33). Indeed, by rendering transplanted HSPCs resistant to antigen-specific depleters, we can replenish the depleted cell subsets (e.g CD123+ or CD33+ cells) without risking to eradicate them if the need for retreatment arises due to minimal residual disease (MRD) leading to relapse. Nevertheless, focusing solely on CD123 or CD33 limits this strategy to specific cell subtypes. It would therefore be desirable to develop a similar system for a cell surface marker present on all hematopoietic cells, such as CD45. Engineering a CD45 variant shielded from antigen-specific depleters would enable protection of the entire blood system post-HCT while still permitting eradication of all CD45+ cells (e.g. host’s blood cells and hematological cancer cells). In this study, we screened CD45’s extra-cellular domain regions with base editors and generated multiple CD45 protein variants altering the binding of antibodies. We selected the CD45-K352E/G variant profile and improved its base editing rate as it showcased loss of binding of a unique anti-CD45 antibody while still maintaining the surface marker’s expression, stability, and function in human hematopoietic stem and progenitor cells (HSPCs). The resulting loss of antibody binding prompted the modification and humanization of the said anti-CD45 antibody, culminating in the development of an anti-CD45 antibody- drug conjugate (CD45-ADC; CIM053-SG3376). Transplantation of the base edited HSPCs into immunodeficient mice showcased their long-term engraftment and multi-lineage reconstitution ability in sequential host mice. Importantly, in AML mouse models xenografted with HSPCs, administration of the CD45-ADC selectively depleted human leukemia and HSPCs-wt cells while preserving the healthy hematopoietic system derived from the transplanted base edited HSPCs. This approach of generating de novo antigens through gene editing to evade targeted-killing represents a robust strategy for creating cell-specific antigens, address the current limitations of HCT but also suggests broader implications beyond hematological malignancies, offering a promising avenue for future therapeutic development.