Clonal dynamics, tolerance, and adverse events after CD45-ADC-conditioned autologous HSPC transplantation in macaques

Compared with total body irradiation (TBI) or chemotherapy, antibody-drug conjugates (ADCs) offer a more targeted and potentially less toxic method for transplantation conditioning. CD45-ADC targets a pan-leukocyte antigen expressed on hematopoietic stem and progenitor cells (HSPCs) and immune cells, offering a promising strategy for gene therapy or allogeneic transplantation. We evaluated reconstitution, clonal dynamics, immune tolerance, and toxicity after conditioning with a DGN549-C-conjugated CD45-ADC followed by autologous transplantation in rhesus macaques. Two doses (0.2 and 0.3 mg/kg) were tested, with HSPC infusion 10 days after conditioning. All animals received barcoded, copepod green fluorescent protein (CopGFP)-expressing lentivirally transduced HSPCs. Both doses resulted in profound depletion of HSPCs and expected cytopenias, with incomplete lymphocyte depletion. With 0.2 mg/kg CD45-ADC conditioning, 2 animals showed robust multilineage engraftment with gene-modified cells and high clonal diversity, comparable with TBI. However, CopGFP+ cell levels and clonal diversity declined at 4 to 8 months, accompanied by development of anti-CopGFP antibodies, suggesting immune rejection. Incomplete T-cell depletion may have contributed. Notably, this rejection was slower and less complete than that after busulfan conditioning, suggesting partial immune tolerance. Dexamethasone treatment in 1 animal reversed rejection and stabilized CopGFP+ levels for >2 years. At 0.3 mg/kg CD45-ADC, 2 animals developed severe respiratory distress 4 to 6 days after transplantation, requiring humane euthanasia, accompanied by elevated inflammatory cytokines. This severe syndrome was not seen in 9 additional animals conditioned with CD45-ADC. These findings highlight the importance of preclinical evaluation of experimental therapeutics. Combining lower-dose CD45-ADC with immune suppression may enable durable engraftment in settings of alloantigen or neoantigen expression.