CD96 Antibody Based Elimination of AML Leukemic Stem Cells As a New Strategy in Stem Cell Transplantation

In AML patients despite intensive chemotherapy and additional stem cell transplantation, residual leukemic stem cells (LSC) may lead to relapse. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently CD96 was identified as marker antigen on AML LSC (Hosen et al., PNAS 104: 11008, 2007). Here, strategies for engineering autologous stem cell grafts as well as for in vivo targeting of residual AML stem cells by addressing CD96 for magnetic cell sorting (MACS) or antibody dependent cellular cytotoxicity (ADCC) are described. To evaluate the efficacy of purging LSC by MACS technology, stem cell containing grafts were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-Biotin-microbeads (Miltenyi) up to a 1000-fold depletion of targeted cells was achieved. Viability, cell count and the potential of HPC to proliferate and differentiate were not affected by this procedure as shown by flow cytometry and colony forming assays. Eradication of AML stems cells is also an issue after allogeneic stem cell transplantation. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1 Fc were generated. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 µg/ml vs. 2 µg/ml). Moreover, also NK cell mediated lytic properties against CD96-positive target cells were elevated (EC50: 0.02 µg/ml vs. 0.15 µg/ml) as analyzed in standard ADCC assays. Thus, the purging strategy may be beneficial for the development of graft-engineering strategies to avoid transplantation of AML-LSC and revitalize autologous stem cell transplantation in this indication. The in vivo application may possibly open additional therapeutic avenues in eliminating residual disease in autologous as well as allogeneic situations.