Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.

METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.

RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4⁺and CD8⁺ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.

CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease.