Tracking clonal dynamics, tolerance, and adverse events after CD45-ADC–Conditioned autologous HSPC transplantation in rhesus macaques

Abstract Compared to total body irradiation (TBI) or chemotherapy, antibody-drug conjugate (ADC)–based transplant conditioning offers a more targeted and potentially less toxic approach to clearing bone marrow niches and enabling robust hematopoietic stem and progenitor cell (HSPC) engraftment. Targeting CD45, a pan-leukocyte antigen expressed on both HSPCs and immune cells, is particularly promising for conditioning prior to HSPC gene therapies or allogeneic transplantation, as it may mitigate immune rejection of neo- or allo-antigens, in contrast to ADCs targeting KIT (CD117). We evaluated engraftment, clonal dynamics, immune tolerance, and clinical parameters following CD45-ADC–conditioned autologous transplant in rhesus macaques (RMs). Two dosing regimens of CD45-ADC were tested. Two RMs (HAWX and H64K) received a single dose of 0.2 mg/kg CD45-ADC and two (DI8F and AR-622) received 0.3 mg/kg CD45-ADC 10 days prior to reinfusion (day 0) of previously collected CD34+ HSPCs transduced with a CopGFP-expressing and barcoded lentiviral vector. All animals exhibited marked HSPC depletion, neutropenia, thrombocytopenia and anemia, with incomplete blood lymphocyte depletion compared to historical experience with TBI. Flow cytometry showed partial depletion of blood and lymph node T cells at both doses on the day of transplantation. No other acute clinical or laboratory abnormalities were observed after CD45-ADC and before transplantation in either dose group. Both 0.2 mg/kg animals recovered normal blood counts and initially demonstrated high-level, multilineage engraftment of GFP+ barcoded cells for 4-8 months, with levels in myeloid cells of 25 to 67% and lower levels in T cells, comparable to TBI- and busulfan-conditioned RMs. Barcode tracking revealed highly polyclonal, multilineage and diverse engraftment during this period, similar to animals transplanted following TBI or busulfan. However, clone number, clonal diversity, and %GFP+ cells began to decline at 8 months (H64K) and 4 months (HAWX) post-transplant, coinciding with the development of anti-GFP antibodies, suggesting immune rejection of CopGFP-expressing cells. Notably, the rejection was slower and less complete than that observed following busulfan conditioning, indicating partial tolerance induced by CD45-ADC. HAWX was treated with dexamethasone (DEX) months 5–7 post-transplant in response to signs of rejection, with arrest of the decrease in vector-containing cells and rescue of multilineage polyclonality, sustained to 27 months, even with discontinuation of DEX. Both animals receiving the higher dose 0.3 mg/kg CD45-ADC developed severe clinical signs and symptoms 4–6 days after cell infusion, including respiratory distress, greatly elevated c-reactive protein levels indicating acute inflammation, and elevated pro-inflammatory cytokines, requiring humane euthanasia within a day of the onset of these clinical abnormalities. All microbial cultures for both animals were negative. Necropsy findings on DI8F and AR-622 revealed acute pulmonary fibrinous edema with necrosis and hemorrhage. The marrow was as expected hypocellular, while lymph nodes had structure preserved and normal cellularity. Although animals conditioned with TBI or busulfan did not exhibit this clinical constellation in our historical experience, 3 of 10 animals receiving 0.2-0.3 mg/kg of CD45-ADC followed by transplant of gene edited CD34+ HSPCs (Demirci, et al. Cell Stem Cell, 2025) showed similar but less severe clinical signs. Cytokine and chemokine profiling of both DI8F and AR-622 showed elevated inflammatory cytokines IL-6, IL-1β, and TNFα. In DI8F, increased levels of CXCL9, CXCL11, and IL-2 were also observed, along with elevated TNF, VEGFA, and MMP12, suggesting systemic inflammation and possible endothelial involvement. AR-622 had lower absolute levels of these cytokines, possibly due to corticosteroids begun prophylactically starting on day -2 until the day of death. Animals with milder clinical signs also had elevations of many of the same cytokines/chemokines following CD45-ADC and transplantation. These findings highlight the need for dose optimization and investigation of the mechanism of these clinical events. Combining immunosuppression with CD45-ADC may provide an effective and targeted strategy to enable engraftment of genetically-modified or allogeneic HSPCs.