Immunotoxin Enables Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) can cure non-malignant hemoglobinopathies, congenital immunodeficiencies and, perhaps, AIDS, diseases that affect millions of people worldwide; yet, it's use is limited outside of malignancies largely because allogeneic transplantation can be complicated by serious conditioning-related toxicities and graft-versus-host disease (GVHD). The advent of highly efficient gene editing technology promises to enable autologous HSCT while avoiding GVHD. However, conditioning-related toxicities will remain a significant barrier to patient accrual unless addressed.

Current conditioning approaches rely on genotoxic methods such as total body irradiation (TBI) and/or chemotherapy. These non-targeted methods induce cytopenias, adversely affect niche cells and may damage other organ systems including germ cells. To fully realize the curative potential of HSCT, the development of mild-conditioning methods that preserve immunity while enabling efficient donor engraftment is essential. Efforts to develop mild non-genotoxic approaches (e.g. CD117 antagonist ACK2 antibody) have thus far been met with efficacy limited to immunocompromised animals with very poor engraftment in immunocompetent settings.

While extensively explored in cancer therapy, protein-based internalizing immunotoxins (ITs) that induce cell death by inhibiting protein synthesis have not been explored as a conditioning strategy. By creating saporin-based ITs against various antigens present on HSCs, we identified an immunotoxin against CD45 receptor (CD45-IT) that potently depleted hematopoietic cells in vitro (40-70 picomolar IC₅₀) and achieved efficient HSC depletion (>98% depletion in vivo)in fully immunocompetent mice when administered as a single dose. Following CD45-IT administration, transplantation of whole bone marrow donor cells enabled efficient donor engraftment (>90% chimerism). A wide engraftment window was tolerated as transplantation anywhere between 2-12 days post CD45-IT resulted in equivalent engraftment. Immune reconstitution was multi-lineage, durable (maintained for >15 months) and transferrable to secondary recipients, indicative of true stem cell engraftment. Using sickle cell disease mice, we demonstrated CD45-IT conditioning enabled >90% healthy donor cell engraftment resulting in complete disease correction (n=18 sickle mice).

We next compared the toxicities induced by CD45-IT and conventional TBI (sub-lethal) in non-transplanted mice over a 120-day period. CD45-IT was non-lethal as mice survived long-term without requiring transplantation (n=12 mice). In contrast to TBI, CD45-IT had significantly less adverse effects on marrow hematopoietic progenitors, vascular integrity and overall marrow cellularity, which recovered faster than in TBI-treated mice. While peripheral B and T-cells were efficiently depleted by both CD45-IT and TBI immediately after treatment, unusually rapid lymphocyte recovery was observed following CD45-IT (12-18 days for T- and B-cell recovery) in comparison to TBI (48 days for recovery). Histology of thymi revealed CD45-IT does not induce thymic atrophy (unlike TBI) as the cortical region continued to support T-cell development as confirmed by T-cell receptor excision circle (TREC) analysis.

Whereas TBI reduced peripheral neutrophil counts versus control, CD45-IT induced a rapid expansion of neutrophils (3-fold higher than naïve control). To test innate immunity, we performed a systemic challenge of candida albicans (a clinically relevant fungal strain). Candida challenge in TBI-conditioned mice proved lethal (100% death in 3 days, p value < 0.0001, n=10 mice). In contrast, CD45-IT treated mice were significantly immune to candida challenge with comparable survival to naïve controls (60% survival for CD45-IT, n=10 mice per group).

To summarize, a protein-based immunotoxin targeting CD45 enables efficient HSCT in fully immunocompetent animals. The targeted immunotoxin avoids numerous toxicities associated with current genotoxic conditioning approaches and preserves innate immunity, thymic integrity and enables quicker recovery of adaptive immune cells while avoiding loss of vascular integrity, undesirable anemia, and prolonged cytopenias. Given these advantages, CD45-IT may be useful for conditioning of non-malignant transplant patients.