Non-Genotoxic Conditioning Using Amanitin Antibody-Drug Conjugates Targeting CD45 Effectively Deplete Human and Non-Human Primate Hematopoietic Stem Cells and Immune Cells

Introduction Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. To safely condition patients for BMT, we are developing antibody drug conjugates (ADCs) targeting CD45, a target expressed throughout the hematopoietic system to specifically deplete both hematopoietic stem cell (HSCs) and immune cells prior to transplant. Results We created anti-CD45 ADCs by conjugating antibodies to alpha-amanitin payload (an RNA polymerase II inhibitor). A resulting anti-CD45 amanitin ADC (CD45-AM) exhibited potent in vitro killing of primary human bone marrow CD34+CD90+ HSCs (IC50 67 pM, Figure 1A ) and PBMC immune cells (IC50 55 pM, Figure 1B ). Next, we explored the CD45-AM ADC in vivo in humanized NSG mice. A single dose of 3 mg/kg CD45-AM enabled >95% depletion of human CD34+ cells in the bone marrow ( Figure 1C ) and >95% depletion of human B-, T- and myeloid cells in the periphery and bone marrow. Control non-targeting isotype matched-ADCs and anti-CD45 antibody not bearing a toxin had minimal effect on either HSC or immune cells. As CD45 is highly expressed on many leukemia and lymphomas, we also assessed the leukemia-depleting activity of CD45-AM in an ALL model and 3 PDX-AML models. A single dose of 1 mg/kg CD45-AM more than doubled the median survival in all 4 models ( p values As BMT will require fast clearing ADCs to avoid depleting the incoming graft, we engineered a fast half-life CD45-AM variant with a half-life of 8-15 hours in mice, and 9 hours in cynomolgus monkeys. Dose escalation of the cross-reactive fast half-life CD45-AM in cynomolgus monkeys showed dose-dependent peripheral lymphocyte depletion. A single dose of 1 mg/kg was well tolerated and enabled >95% depletion of HSCs (CD34+CD90+CD45RA- cells) and >80% lymphocyte depletion in the marrow 14 days post-administration ( Figure 1D ). Conclusion Targeting CD45 with an amanitin ADC resulted in potent in vitro and in vivo human HSC and immune cell depletion. The anti-CD45 amanitin ADC significantly reduced disease burden in multiple leukemia models. Preliminary assessment of a fast half-life ADC in cynomolgus monkeys showed efficient HSC and immune cell depletion in the marrow. An alpha-amanitin ADC targeted to CD45 may i) be non-genotoxic; ii) avoid bystander toxicity, due to amanitin's poor cell permeability as a free toxin; and iii) kill cycling and non-cycling cells, the latter being necessary for effective HSC and immune cell depletion. Together, these properties may enable safer targeted conditioning and expand the use of BMT in malignant and non-malignant conditions.